CN111971298A - Antibodies - Google Patents

Antibodies Download PDF

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Publication number
CN111971298A
CN111971298A CN201980025412.1A CN201980025412A CN111971298A CN 111971298 A CN111971298 A CN 111971298A CN 201980025412 A CN201980025412 A CN 201980025412A CN 111971298 A CN111971298 A CN 111971298A
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China
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seq
antibody
sequence
region
binding
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CN201980025412.1A
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Inventor
D.萨丁
E.C.W.布雷杰
B.E.C.G.德戈伊
K.坎珀
P.恩格尔伯茨
E.N.范登布林克
R.拉德马克
D.沃齐尔
S.霍尔巴赫
P.帕伦
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Genmab AS
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Genmab AS
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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Abstract

The present invention relates to antibodies that bind 5T4, including bispecific antibodies that bind 5T4 and CD 3. The invention further provides pharmaceutical compositions comprising the antibodies, and the use of the antibodies in therapeutic and diagnostic procedures, particularly in cancer therapy.

Description

Antibodies
Technical Field
The present invention relates to antibodies that bind 5T4, including bispecific antibodies that bind 5T4 and CD 3. The invention further provides pharmaceutical compositions comprising the antibodies, and the use of the antibodies in therapeutic and diagnostic procedures, particularly in cancer therapy.
Background
5T4 (also known as trophoblast glycoprotein TPBG or Wnt-activated inhibitor 1 WAIF 1) is a 72kDa single-pass transmembrane protein containing 8 leucine-rich repeats (LRR) and 7 potential N-glycosylation sites (Zhao et al, 2014Structure 22, 612-.
In addition to placenta, 5T4 expression was restricted in normal adult tissues (Southall et al, 1990Br J Cancer 61, 89-95). 5T4 is expressed in a number of human cancers, including renal, cervical, ovarian, lung, prostate and colon cancers (Stern and Harrop,2017Cancer Immunol Immunother 66, 415-426; Southall et al, 1990Br J Cancer 61, 89-95). Expression of 5T4 in tumor cells promotes epithelial to mesenchymal transformation by 1) (Damelin et al, 2011Cancer Res 71, 4236-; carsberg et al, 1996Int J Cancer 68,84-92), and 2) inhibition of the canonical Wnt/beta-catenin signaling pathway and activation of the non-canonical Wnt pathway (Kagermeier-Schenk et al, 2011Dev Cell 21, 1129-.
The 5T 4-targeted antibody and 5T 4-targeted therapy have clinical activity in several cancers known to express 5T4, including colorectal, lung and renal cancers. For example, naptumomab estafenatox is a recombinant fusion protein consisting of a 5T4-Fab portion genetically fused to an engineered superantigen variant SEA/E-120. Currently, it is in clinical trials as an immunotherapy for non-small cell lung cancer (NSCLC), renal cells (RCC) and pancreatic cancer (see, e.g., Eisen, et al, 2014Curr Oncol Rep 16,370). In addition to this, the present invention is,
Figure BDA0002720035660000011
is a modified vaccinia Ankara expressing the 5T4 construct (MVA-5T4) that shows clinical benefit in colorectal, prostate and renal cancers (see, e.g., Stern and Harrop,2017Cancer Immunol Immunother 66, 415-426; Scrurr et al, 2017JAMA Oncol 12, 10). Other anti-5T 4 antibodies are described in WO2007106744, WO03038098, WO2011048369, WO2013041687, WO 2017072207.
Despite major advances in eradication of cancer, there remains a need for further improvements in antibody-based cancer therapies.
Summary of The Invention
It is an object of the present invention to provide an antibody comprising at least one antigen binding region capable of binding 5T4 (trophoblast glycoprotein), wherein said antibody is capable of blocking the binding to 5T4 of an antibody [059] comprising a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID NO:5 and a light chain Variable (VL) region comprising the sequence set forth in SEQ ID NO: 9.
The antibody may particularly be a bispecific antibody and may further comprise an antigen binding region of an antibody that binds CD3, such as human CD3(epsilon), human CD3(epsilon) as specified in SEQ ID NO: 4.
In another aspect, the invention relates to a nucleic acid construct comprising
a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined herein, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined herein.
In another aspect, the present invention relates to an expression vector comprising
a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined herein, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined herein.
In another aspect, the invention relates to a cell comprising a nucleic acid construct or expression vector as defined herein.
In another aspect, the invention relates to a composition comprising an antibody according to the invention.
In another aspect, the invention relates to a pharmaceutical composition comprising an antibody as defined herein and a pharmaceutically acceptable carrier.
In another aspect, the invention relates to an antibody as defined herein for use as a medicament, such as for the treatment of a disease.
In another aspect, the invention relates to a method of treating a disease or disorder, the method comprising administering to a subject in need thereof an antibody, composition or pharmaceutical composition as defined herein.
In another aspect, the invention relates to a method for producing an antibody as defined herein.
In another aspect, the invention relates to a kit comprising an antibody as defined herein; and instructions for use of the kit.
In another aspect, the invention relates to an anti-idiotype antibody that binds to an antigen binding region of an antibody as defined herein that is capable of binding to 5T 4.
Brief Description of Drawings
FIG. 1: IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR used in combination with IgG1-5T4-A3-F405L and IgG1-5TAntibody replacement of 4-226-FEAR. Antibody displacement (displacement) was determined by biolayer interferometry on an Octet HTX instrument (ForteBio). IgG1-5T4-A3-F405L was immobilized on the biosensor and loaded with human 5T4ECDHI (mature protein of SEQ ID NO. 99). Subsequently, the loaded biosensor was exposed to IgG1-5T4-A3-F405L, IgG1-5T4-H8-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T 4-226-FEAR. The figure shows the association response (500s) after exposure to a second antibody. A-C. IgG1-5T4-A3-F405L showed no binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHI complex, indicating cross-blocking (self-blocking) with IgG1-5T 4-A3-F405L. The IgG1-5T4-H8-FEAR antibody showed increased mass (indicating binding to the immobilized IgG1-5T4-A3-F405L-5T4ECDHI complex) and thus was not cross-blocked with IgG1-5T 4-A3-F405L. IgG1-5T4-059-FEAR, b.igg1-5T4-207-FEAR and c.igg1-5T4-226-FEAR all showed an initial increase in mass (indicating binding of the antibody to the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex) followed by a rapid decrease in mass. This behavior of the antibody is indicative of an antibody substitution (Abdiche YN, et al (2017) Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can display One animal plos ONE 12(1): e0169535.doi:10.1371/journal. po. 0169535).
FIG. 2: simultaneous binding of 5T4 antibody to membrane bound 5T4 as measured by flow cytometry.5T4 antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR were conjugated to Fluorescein Isothiocyanate (FITC) and added to 5T 4-expressing SK-OV-3 cells at a concentration of 2 μ g/mL in the presence of 10 μ g/mL of unconjugated IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12, IgG1-5T4-207-FEAR or IgG1-5T 4-226-FEAR. The percent binding of FITC-labeled antibodies was calculated and plotted as the mean percent binding ± Standard Deviation (SD).
FIG. 3: binding of the 5T4 antibody to HEK-293 cells transfected with full-length human and chicken 5T 4.HEK-293 cells transiently transfected with full-length human 5T4(SEQ ID NO:1) (A) or chicken 5T4(SEQ ID NO:3) (B) were incubated with various concentrations of IgG1-5T4-A3-F405L, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR antibodies. To conjugation withAfter incubation with goat anti-human IgG F (ab')2 with R-Phycoerythrin (PE), the Mean Fluorescence Intensity (MFI) was determined by flow cytometry. As a negative control, IgG1-b12-K409R (10. mu.g/mL) was included.
FIG. 4: internalizing ability of the monovalent 5T4 antibody.Toxin-conjugated bispecific antibodies were generated by controlled Fab arm exchange of unconjugated 5T4 antibody with an IgG1-b12 antibody (HIV-1gp120 specific) each of which has been conjugated to one Duostatin-3 molecule, which antibody recognizes 5T4 with one Fab arm and an unrelated antigen (HIV-1gp120, which is not expressed on tumor cells) with a second Fab arm. As indicated, MDA-MB-468(A) and HCC1954(B) cells were incubated with increasing concentrations of antibody. Cell viability was measured after 5 days. Data are expressed as the average percentage of viable cells of three replicates. As a negative control, monospecific bivalent IgG1-b12(IgG1-b12-vcDuo3) conjugated to Duostatin-3 was included.
Fig. 5 (I): CD3x5T4 bispecific antibodies and full-length human and cynomolgus monkey transfected into HEK-293 cells (cynomolgus monkey)5T 4.Binding of monovalent and bivalent 5T4 antibodies was analyzed using HEK-293 cells transiently transfected with either full-length human (left panel) or cynomolgus monkey 5T4 (right panel). Cells were incubated with increasing concentrations of antibody as indicated. After secondary labeling with FITC-conjugated goat anti-human IgG F (ab')2, binding was analyzed by flow cytometry. As a negative control antibody, IgG1-b12-K409R (3. mu.g/mL) was included. Data are presented as Mean Fluorescence Intensity (MFI) values ± SD of two technical replicates. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR. Binding of bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T 4-H8-FEAR.
Fig. 5 (II): binding of bispecific CD3x5T4 antibody to cynomolgus monkey and human 5T4 transfected into HEK-293 cells. The monovalent and bivalent binding of the 5T4 antibody was analyzed using HEK-293 cells transiently transfected with human 5T4 (left panel) or cynomolgus monkey 5T4 (right panel). Cells were incubated with increasing concentrations of antibody as indicated. After secondary labeling with Phycoerythrin (PE) -conjugated goat anti-human IgG F (ab')2, binding was analyzed by flow cytometry. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T 4-106-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR and IgG1-5T 4-076-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T 4-085-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T 4-127-FEAR;
Binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T 4-A1-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T 4-A3-FEAR.
Fig. 6 (I): binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to 5T4 positive human tumor cells.Monovalent and bivalent binding of 5T4 antibody to HeLa cells (left panel) or MDA-MB-231 cells (right panel) was determined by flow cytometry. Cells were incubated with increasing concentrations of antibody. MFI was determined by flow cytometry after secondary labeling with FITC-conjugated goat anti-human IgG F (ab') 2. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right panel). IgG1-b12-K409R (3. mu.g/mL) was included as a negative control (open circles). Fig. 6 (II): binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to HeLa cells. Monovalent and bivalent binding of 5T4 antibody to HeLa cells was determined by flow cytometry. Cells were incubated with increasing concentrations of antibody. After secondary labeling with Phycoerythrin (PE) -conjugated goat anti-human IgG F (ab')2, the Mean Fluorescence Intensity (MFI) was determined by flow cytometry.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR;
binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T 4-106-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T 4-085-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T 4-127-FEAR;
binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T 4-A1-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR
Fig. 6 (III): binding of CD3x5T4 bispecific and 5T4 monospecific antibodies to MDA-MB-231 cells. Monovalent and bivalent binding of 5T4 antibody to MDA-MB-231 cells was determined by flow cytometry. Cells were incubated with increasing concentrations of antibody. After secondary labeling with goat anti-human IgG F (ab') 2 conjugated with PE, the Mean Fluorescence Intensity (MFI) was determined by flow cytometry. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T 4-106-FEAR;
Binding of bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T 4-085-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T 4-127-FEAR; binding of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T 4-A1-FEAR;
binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T 4-A3-FEAR.
Fig. 7 (I): CD3X5T4 bispecific antibody in MDA-MB-231 cells using purified T cells as effector cells Cytotoxicity was induced in vitro in cells.MDA-MB-231 cells were incubated with increasing concentrations of either CD3x5T4 bispecific antibody or monospecific bivalent 5T4 antibody and isolated T cells as effector cells in an effector to target (E: T) ratio of 8: 1. Purified T cells obtained from two different donors were used in this experiment,donor a (left panel) and donor B (right panel). Cytotoxicity was determined by measuring the percentage of live MDA-MB-231 cells after 72 hours incubation (% live cells ═ sample absorbance-absorbance of staurosporine treated target cells]/[ absorbance of untreated target cells-absorbance of staurosporine-treated target cells]x 100)。
A. In bsIgG1-huCD3-FEALx5T4-207-FEAR,
cytotoxicity induced in the presence of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; B. cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; C. cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR.
Fig. 7 (II): CD3X5T4 bispecific antibody in MDA-MB-231 cells using purified T cells as effector cells IC50 value for in vitro induced cytotoxicity in cells. The MDA-MB-231 cells were analyzed for bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR using GraphPad Prism V7.02 software,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR or
IC50 values for bsIgG1-huCD3-H101G-FEALx5T 4-059-FEAR-induced T cell-mediated cytotoxicity. Data are presented as mean IC50 values ± SD of two different donors.
Fig. 8 (I): the CD3x5T4 bispecific antibody induced cytotoxicity in MDA-MB-231 cells using T cells as effector cells in vitro. MDA-MB-231 cells were incubated with increasing concentrations of CD3x5T4 bispecific antibody or 5T4 homodimer and isolated T cells as effector cells at an E: T ratio of 8: 1. Three different donors were used for this experiment. Data shown are the mean% survival ± standard error of the mean (SEM) of the three donors tested.
A. In bsIgG1-huCD3-FEALx5T4-207-FEAR,
t cell-mediated cytotoxicity (decreased survival) induced in the presence of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR;
B. in the case of bsIgG1-huCD3-FEALx5T4-226-FEAR,
T cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; C. t cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR; D. t cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T 4-106-FEAR; E. t cell-mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T 4-A1-FEAR; F. t cell mediated cytotoxicity induced in the presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T 4-A3-FEAR.
Fig. 8 (II): IC50 values for cytotoxicity induced by CD3x5T4 bispecific antibody in MDA-MB-231 cells using T cells as effector cells in vitro. IC50 values of CD3x5T4 bispecific antibody-induced T cell-mediated cytotoxicity in MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are presented as mean IC50 values ± SD of three different donors. IC50 values for T cell mediated cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T 4-A3-FEAR; B. IC50 values for T cell mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T 4-A3-FEAR.
Fig. 9 (I): in vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells.Isolation of MDA-MB-231 cells from increasing concentrations of CD3x5T4 bispecific antibody and monospecific bivalent 5T4 antibody (as indicated) and as effector cellsThe T cells of (1) were incubated together with an E: T ratio of 8: 1. Three T cell activation markers (PD1[ top panel ] were analyzed by flow cytometry]CD25 middle panel]And CD69[ lower panel ]]) Expression of (2). Two different donors were used for this experiment, donor a (filled symbols) and donor B (open symbols). A. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; B. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; C. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T 4-059-FEAR.
Fig. 9 (II): in vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of MDA-MB-231 cells EC50 value.The EC50 values of in vitro T cell activation markers (FEPD 1, CD25 and CD69) induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR in the presence of MDA-MB-231 cells were analyzed using GraphPad Prism V7.02 software. Data are presented as mean ± SD of two different donors.
Fig. 10 (I): in vitro T cell activation by CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells. MDA-MB-231 cells were incubated with increasing concentrations of CD3x5T4 bispecific antibody and 5T4 homodimer and isolated T cells as effector cells at an E: T ratio of 8: 1. By CD4+(left panel) and CD8+(right panel) increase in% CD69+ cells within the T cell population to measure T cell activation. Three different donors were used for this experiment; data shown is mean% CD69 upregulation ± SEM of the three donors tested. A. T cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T 4-207-FEAR; B. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T 4-226-FEAR; C. in bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEARInduced T cell activation in the presence; D. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T 4-106-FEAR; E. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T 4-A1-FEAR; F. t cell activation induced in the presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T 4-A3-FEAR.
Fig. 10 (II): EC activated by T cells in vitro of CD3x5T4 bispecific antibody in the presence of MDA-MB-231 cells50The value is obtained. Analysis of the T cell activation marker induced in vitro by the CD3X5T4 bispecific antibody in the presence of MDA-MB-231 cells using GraphPad Prism V7.02 software (CD 4)+And CD8+CD69 in T cell populations+[A-B],CD25+[C-D]And PD1+[E-F]Increase in CD25 and CD69 cells)%)50The value is obtained. Data are presented as mean ± SD of three different donors. A. EC50 values of CD69 up-regulated by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR, and bsIgG1-huCD3-FEALx5T 4-A3-FEAR;
B. EC CD69 upregulated by induction of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR50A value;
C. EC for CD25 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR 50A value; D. EC CD25 upregulated by induction of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR50A value;
E. prepared from bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR, and bsIgG1-huCD3-FEALx5T 4-A3-FEAR-induced EC 1 upregulation in PD150A value;
F. EC 1 upregulated by PD1 induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR50The value is obtained.
FIG. 11: t-cell cytokines induced by CD3x5T4 bispecific antibodies in the presence of 5T4 positive tumor cells And (4) releasing.MDA-MB-231 cells were incubated with 0.2. mu.g/mL CD3x5T4 bispecific antibody (bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101 3-FEALx5T 3-226-FEAR, bsIgG 3-huCD 3-FEALx5T 3-059-FEAR or bsIgG 3-CD 3-H101-FEALx 5T 3-059-FEAR) and 5T 3 monospecific antibody (IgG 3-5T 3-FEAR, IgG 36207-FEAR, IgG 3-H101-FEALx 5T 3-059-FEAR or IgG 3-FEAR) as effector ratio cells and the MDA 3-FEAR was isolated. Cytokine release was analyzed by U-PLEX assay. A. Concentrations of IL-10, IL-13 and TNF in supernatants of T cell (donor A-derived) tumor cell co-cultures after 72 hours incubation with either CD3x5T4 bispecific antibody or 5T4 monospecific antibody. B. Concentrations of IL-10, IL-13 and TNF in supernatants of T cell (donor B-derived) tumor cell co-cultures after 72 hours incubation with either CD3x5T4 bispecific antibody or 5T4 monospecific antibody.
FIG. 12: t ratio of different E using PBMC as effector cells, bispecific antibody by CD3x5T4 at SK- Cytotoxicity was induced in OV-3 cells in vitro.SK-OV-3 cells were incubated with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right panel) and PBMC as effector cells at E: T ratios of 1:2, 1:1, 2:1, 4:1, 8:1 and 12: 1. Cytotoxicity was determined by measuring the percentage of live SK-OV-3 cells after 72 hours incubation (% live cells ═ sample absorbance-staurosporine treated targetAbsorbance of the cells]/[ absorbance of untreated target cells-absorbance of staurosporine-treated target cells]]x 100). PBMCs from two different donors were used in this experiment: A. donor C and b.
FIG. 13: t cells used as effector cells at different E: T ratios from CD3x5T4 bispecific antibodies at SK- Cytotoxicity was induced in OV-3 cells in vitro.SK-OV-3 cells were incubated with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR (left panel) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right panel) and isolated T cells as effector cells at E: T ratios of 1:2, 1:1, 2:1, 4:1 and 8: 1. Cytotoxicity was determined by measuring the percentage of live SK-OV-3 cells after 72 hours incubation (% live cells ═ sample absorbance-absorbance of staurosporine treated target cells ]/[ absorbance of untreated target cells-absorbance of staurosporine-treated target cells]]x 100). PBMCs from two different donors were used in this experiment: A. donor E and b.
FIG. 14 CD3x5T4 bispecific antibody in the MDA-MB-231 xenograft model in NSG-HIS mice The antitumor activity of (1).A. Mean tumor size in MDA-MB-231 xenograft model in NSG-HIS mice after treatment with PBS (vehicle control), 0.5mg/kg bsIgG1-huCD3-FEALx5T4-207-FEAR or 0.5mg/kg bsIgG1-huCD3-H101G-FEALx5T 4-207-FEAR. Tumor size was assessed by caliper measurements. Error bars indicate SEM. B. After treatment with PBS, bsIgG1-huCD3-FEALx5T4-207-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, the tumor size was determined<500mm3Percentage of NSG-HIS mice injected with MDA-MB-231 cells.
FIG. 15: binding of direct FITC-labeled 5T 4-specific antibody to human 5T4 variant having a single alanine mutation at positions 32 to 355 of human 5T4 ECD, as determined by flow cytometry. Binding was expressed as Z-score (fold change) as a measure of change in binding compared to the non-cross-blocking 5T 4-specific control antibody used for normalization (bsIgG1-5T4-a1-F405Lxb 12-FEAR-FITC). The numbers on the x-axis refer to the amino acid position in human 5T4(SEQ ID: 1). Residues with a Z-score of binding below-1.5 (indicated by the dashed line) are considered "binding-loss variants". Residues with positive Z scores in binding were non-cross-blocking binding-loss residues of a 5T 4-specific control antibody (bsIgG1-5T4-A1-67F 405Lxb 12-FEAR-FITC). The residues at aa positions 38,45,49,51,54,62,64,66,68,71,72,77,91,104,108,110,112,118,121,122,135,137,155,161,167,171,201,202,205,208,218,231,269,279,298,300,303,323,324,340 and 344 are not evaluated because these positions contain endogenous alanine or cysteine. Data shown are Z scores for binding of (A) bsIgG1-B12-FEALx5T4-059-FEAR-FITC, (B) bsIgG1-B12-FEALx5T4-207-FEAR-FITC, (C) bsIgG1-B12-FEALx5T4-226-FEAR-FITC, and (D) bsIgG1-5T4-A3-F405Lxb 12-FEAR-FITC. Z scores well below-1.5 and buried residues predicted to be spatially separated from most surface-exposed binding-loss residues are excluded (for bIgG1-b12-FEALx5T 4-207-FEAR-FITC: L281[ Z score: -1.57] and P326[ Z score: 1.54; for bsIgG1-b12-FEALx5T 4-226-FEAR-FITC: L273[ Z score: -1.58], L281[ Z score: -1.65], N294[ Z score: -1.57], L309[ Z score: -1.63] and P326[ Z score: -1.67 ]).
Fig. 16 (I): cytotoxicity was induced in vitro by the CD3x5T4 bispecific antibody in tumor cells of different indications using T cells as effector cells. Tumor cells were incubated with increasing concentrations of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibody (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) and isolated T cells as effector cells in the E: T ratio of 4: 1. Cytotoxicity (decreased survival) was determined by measuring the percentage of viable tumor cells after 72 hours of incubation. Data shown are mean% survival ± SEM of duplicate wells from one representative donor of at least three tested donors. A. Cytotoxicity induced in pancreatic cancer cell lines (decreased survival); B. cytotoxicity (decreased survival) induced in cervical cancer cell lines.
Fig. 16 (II): IC50 values of cytotoxicity induced in vitro by CD3x5T4 bispecific antibody in tumor cell lines of different indications using T cells as effector cells. IC50 values for T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in tumor cells for indicated indications were analyzed using GraphPad Prism V7.02 software. Data are presented as mean IC50 values (see table 10) ± SD for at least three different donors.
Fig. 17 (I): in vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of tumor cells of different indications. Tumor cells were incubated with increasing concentrations of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibody (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) and isolated T cells as effector cells for 72 hours at an E: T ratio of 4: 1. By CD4+(left panel) and CD8+(right panel) upregulation of CD69(CD69+ cell%) within the T cell population to measure T cell activation. Data shown are mean% CD69+ cells ± SD from duplicate wells of one representative donor of at least three tested donors. A. T cell activation induced by CD3x5T4 bispecific antibody in the presence of pancreatic cancer cell line BxPc-3; B. t cell activation induced by a CD3x5T4 bispecific antibody in the presence of pancreatic cancer cell line PANC-1; C. t cell activation induced by a CD3x5T4 bispecific antibody in the presence of a cervical cancer cell line SiHa; D. t cell activation induced by a CD3x5T4 bispecific antibody in the presence of a cervical cancer cell line Ca Ski.
Fig. 17 (II): EC50 values for in vitro T cell activation by CD3x5T4 bispecific antibodies in the presence of tumor cell lines of different indications. Analysis of the EC50 values for T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR co-cultured with tumor cell lines of different indications (CD 4) using GraphPad Prism V7.02 software +And CD8+CD69+ cells within T cell population). Data are presented as mean EC50 values (see table 10) ± SD for at least three different donors. A. EC50 values for CD4+ T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of indicated tumor cell lines; B. EC50 values for CD8+ T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of an indicator tumor cell line.
Detailed Description
Definition of
As used herein, the term "antibody" (Ab) is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any thereof, that has the ability to specifically bind to an antigen under typical physiological and/or tumor-specific conditions for a half-life of a substantial period of time, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period of time (e.g., a time sufficient to induce, promote, enhance and/or modulate a physiological response associated with binding of an antibody to an antigen and/or a time sufficient for the antibody to be internalized). Binding regions that interact with an antigen (or binding domains having the same meaning as may be used herein) include the variable regions of the heavy and light chains of an immunoglobulin molecule. The constant region of an antibody (Ab) may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system (e.g., C1q), the first component in the classical pathway of complement activation.
In the context of the present invention, the term "antibody" includes monoclonal antibodies (mabs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, as well as "antibody fragments" or "fragments thereof" provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant techniques, which retain the ability to specifically bind to an antigen (antigen-binding fragment) and retain the ability to conjugate to a toxin. Unless the disclosure herein is otherwise limited, antibodies defined according to the present invention may be of any isotype.
As indicated above, the term antibody as used herein includes antibody fragments that retain the ability to specifically interact with, such as bind to, an antigen, unless otherwise indicated or clearly contradicted by context. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of a light chain variable domain (VL), a heavy chain variable domain (VH), a light chain constant region (CL) and a heavy chain constant region 1(CH1) domain or monovalent antibodies as described in WO 2007/059782; (ii) f (ab')2A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting essentially of VH and CH1 domains; (iv) (iv) an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) an Fv fragment consisting essentially of a VH domain, also known as the domain antibody Holt et al; trends biotechnol.2003nov; 21(11) 484-90 dAb fragment Ward et al, Nature341544-; (vi) camelid (camelid) or nanobodies (nanobodies) Revets et al; expert Opin Biol ther.2005jan;5(1) 111-24 and (vii) isolated Complementarity Determining Regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single chain antibody or single chain Fv (scFv), see, e.g., Revets et al; Expert Opin Biol Ther.2005 Jan;5(1) 111-24 and Bird et al, Science242,423-426(1988). Unless otherwise indicated or the context clearly indicates otherwise, such single chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of antibodies, they are, collectively and individually, unique features of the invention, exhibiting different biological properties and utilities. These and other useful antibody fragments are discussed further herein in the context of the present invention.
The antibody may be produced and collected in different in vitro or ex vivo expression or production systems, e.g., from a recombinant modified host cell, hybridoma or system using a cellular extract that transcribes and/or translates a nucleic acid sequence encoding the antibody outside of the support. It will be appreciated that a plurality of different antibodies (which antibodies are as defined in the context of the present invention) may be provided by producing each antibody separately in a production system as described above, and then mixing the antibodies, or by producing several antibodies in the same production system.
As used herein, the term "immunoglobulin heavy chain" or "immunoglobulin heavy chain" refers to one of the heavy chains of an immunoglobulin. Heavy chains generally consist of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that define the immunoglobulin isotype. The heavy chain constant region is typically composed of three domains, CH1, CH2, and CH 3. As used herein, the term "immunoglobulin" means a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four potentially interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology Ch.7(Paul, W., ed.,2 nd. raven Press, N.Y. (1989)). within the structure of an immunoglobulin, two heavy chains are interconnected via disulfide bonds in a so-called "hinge region". As with the heavy chains, each light chain is typically composed of several regions, a light chain variable region (referred to herein simply as VL) and a light chain constant region, the light chain constant region is typically composed of a domain CL CDR1, FR2, CDR2, FR3, CDR3, FR 4. CDR sequences are defined according to IMGT (see Lefranc MP.et al, Nucleic Acids Research,27,209-212,1999] and Brochet X.Nucl.acids Res.36, W503-508 (2008)).
As used herein, the terms "half molecule", "Fab arm" and "arm" refer to a heavy chain-light chain pair. When a bispecific antibody is described as comprising a "derived" from a first antibody and a "derived" from a second antibody, the term "derived" means that the bispecific antibody is produced by recombining the half molecules from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, "recombinant" is not intended to be limited by any particular recombinant method, and thus includes all methods of producing bispecific antibodies, e.g., as described below, including, e.g., recombination by half-molecule exchange as well as recombination at the nucleic acid level and/or recombination by co-expression of two half-molecules in the same cell.
As used herein, the term "antigen binding region" or "binding region" refers to a region of an antibody that is capable of binding an antigen. The antigen may be any molecule, such as a polypeptide, e.g. present on a cell, a bacterium or a virion. Unless the context contradicts, the terms "antigen" and "target" may be used interchangeably in the context of the present invention. Unless the context contradicts, the terms "antigen-binding region" and "antigen-binding site" are used interchangeably in the context of the present invention.
The term "block binding" or "blocking binding of an antibody" or "cross-blocking binding" refers to the situation where binding of one antibody to a particular antigen prevents a second antibody from binding to the same antigen, and vice versa. Each antibody has the ability to bind to an antigen in the absence of the other antibody, as determined by a significant binding response, while one of the antibodies lacks a binding response when the other antibody is present. The ability of one antibody to block the binding of another antibody can be determined in a classical sandwich epitope-box and assay format by biolayer interferometry, for example, as described in example 3 and Abdicche et al (Abdiche YN, Malashock DS, Pinkerton A, Pons J. expanding blocking assays using octets, protein on, and Biacore biosense. anal biochem. 2009; 386(2): 172-. Briefly, in a sandwich epitope box-and-assay, an antibody in solution is tested for binding to its specific antigen, which is first captured by an immobilized antibody. In the context of the present invention, if an antibody is capable of "replacing" another antibody, it does not block the binding of the other antibody, according to the definition of "replacement" below. Unless the context contradicts, the terms "blocking binding" and "blocking binding of antibodies" and "cross-blocking binding" may be used interchangeably in the context of the present invention. Preferably, full length antibodies are used to determine the ability of one antibody to block the binding of another antibody.
The term "displacement" or "displacement capacity" refers to the situation where two antibodies interfere with each other's binding to an antigen by altering each other's binding to its specific antigen through the formation of a transient trimolecular complex that rapidly breaks down by retaining one antibody against the antigen and displacing the other. Antibody substitutions are defined in Abdiche et al, 2017(Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morrishige W, et al (2017) Antibodies Targeting Closel Adjacent or minimal overlaying Epitopes Can One animal ploS ONE 12(1): e0169535.doi: 10.1371/joural. po. 0169535). The displacement of the antibody can be determined by biolayer interferometry in the form of a classical sandwich assay by using a real-time label-free biosensor as described in Abdiche et al 2017 and example 4 of the present application. Preferably, antibody displacement is determined using an antibody in the form of an IgG.
The term "binding" as used herein refers to the binding of an antibody to a predetermined antigen or target, typically as determined by biolayer interferometry using antibodies as ligands and antigens as analytes to correspond to 1E -6M or less, e.g. 5E-7M or less, 1E-7M or less, e.g. 5E-8M or less, e.g. 1E-8M or less, e.g. 5E-9M is less or, e.g., 1E-9K of M or lessDBinds with a binding affinity and with a K corresponding toDBinds to a predetermined antigen, said KDAt least 10-fold lower, such as at least 100-fold lower, such as at least 1000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to non-specific antigens other than the predetermined antigen or closely related antigen (e.g., BSA, casein).
As used herein, the term "KD"(M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, and is determined by the sum of kdDivided by kaAnd (4) obtaining.
As used herein, the term "kd”(sec-1) Refers to the off-rate constant for a particular antibody-antigen interaction. Said value is also referred to as koffValue or off-rate.
As used herein, the term "ka”(M-1x sec-1) Refers to the association rate constant for a particular antibody-antigen interaction. Said value is also referred to as konA value or an association rate.
As used herein, the term "5T 4" refers to the protein named 5T4, which is also known as trophoblast glycoprotein, 5T4 carcinoembryonic antigen, 5T4 carcinoembryonic trophoblast glycoprotein, TPBG, WAIF1, and M6P 1. It is a 72-80kDa transmembrane protein with an extensive N-linked glycosylated core. In humans (Homo sapiens), the 5T4 protein has the amino acid sequence shown as SEQ ID NO:1 (human trophoblast glycoprotein: Uniprot accession Q13641). In the amino acid sequence shown in SEQ ID NO. 1, amino acid residues 1-31 are signal peptides and amino acid residues 32-420 are mature polypeptides. In cynomolgus monkeys (Macaca fascicularis), the 5T4 protein has the amino acid sequence shown by SEQ ID NO:2 (Uniprot accession Q4R8Y 9). In the amino acid sequence shown in SEQ ID NO. 2, amino acid residues 1-34 are signal peptides and amino acid residues 35-420 are mature polypeptides. In chickens (Gallus gallicus), the 5T4 protein has the amino acid sequence shown as SEQ ID NO:3 (Uniprot accession No. R4GM 46). In the sequence shown as SEQ ID NO. 3, amino acid residues 1-27 are signal peptides and amino acid residues 28-379 are mature polypeptides.
As used herein, the term "CD 3" refers to a human cluster of differentiation 3 protein that is part of a T cell co-receptor protein complex and consists of four distinct chains. CD3 is also present in other species, and thus, unless the context contradicts, the term "CD 3" is not limited to human CD 3. In mammals, the complex contains a CD3 gamma (gamma) strand (human CD3 gamma strand UniProtKB/Swiss-Prot No P09693 or cynomolgus monkey CD3 gamma UniProtKB/Swiss-Prot No Q95LI7), a CD3(delta) strand (human CD3UniProtKB/Swiss-Prot No P04234 or cynomolgus monkey CD3 UnitKB/Swiss-Prot No Q95LI8), two CD3(epsilon) strands (human CD3UniProtKB/Swiss-Prot No P07766; amino acid residues 1-22 are signal peptides and amino acid residues 23-207 are mature CD3 polypeptides identified herein as SEQ ID NO: 4; cynomolgus monkey CD3 UnitKB/Swiss-Prot No Q95 zeta 5; or cynomolgus monkey Swiss 3 Swiss-ProtKB/Swiss-ProtKB-ProtNo Q95 zeta # 3663 and cynomolgus monkey CD 6335 (CD 6326) strand (human CD). These chains associate with molecules called T Cell Receptors (TCRs) and produce activation signals in T lymphocytes. The TCR and CD3 molecules together comprise a TCR complex.
The term "antibody binding region" refers to a region of an antigen that comprises an epitope to which an antibody binds. Antibody binding regions can be determined by using the epitope box of biolayer interferometry and by alanine scanning or by shuffling assays (using an antigenic construct that exchanges antigenic regions for one another and determining whether the antibody still binds to the antigen or not). Amino acids within the antibody binding region involved in interaction with an antibody can be determined by hydrogen/deuterium exchange mass spectrometry and by crystallography of the antibody bound to its antigen.
The term "epitope" refers to an antigenic determinant specifically bound by an antibody. Epitopes are typically composed of surface groupings of molecules such as amino acids, sugar side chains, or combinations thereof, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that in the presence of denaturing solvents, binding to the former is lost rather than to the latter. An epitope may comprise amino acid residues directly involved in binding, as well as other amino acid residues not directly involved in binding, such as amino acid residues effectively blocked or covered by the antibody when the antibody binds to an antigen (in other words, amino acid residues within or in close proximity to the footprint of a particular antibody).
As used herein, the terms "monoclonal antibody," "monoclonal Ab," "monoclonal antibody composition," "mAb," and the like refer to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having a variable region and a constant region derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell. Monoclonal antibodies can also be produced from recombinantly modified host cells or systems using cellular extracts that transcribe and/or translate nucleic acid sequences encoding the antibodies outside the support.
As used herein, the term "isotype" refers to the immunoglobulin class encoded by the heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, e.g., IgG1m (za) and IgG1m (f)). Furthermore, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain.
As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent or variant antibody) comprising one or two pairs of heavy and light chains, each heavy and light chain containing all of the heavy and light chain constant and variable domains normally found in heavy-light chain pairs of wild-type antibodies of that isotype. In full-length variant antibodies, the heavy and light chain constant and variable domains may contain, inter alia, amino acid substitutions that improve the functional properties of the antibody, as compared to the full-length parent or wild-type antibody. A full-length antibody according to the present invention can be produced by a method comprising the steps of: (i) cloning the CDR sequences into a suitable vector comprising the complete heavy chain sequence and the complete light chain sequence, and (ii) expressing the complete heavy and light chain sequence system in a suitable expression system. It is within the knowledge of the person skilled in the art to generate full-length antibodies when starting from a CDR sequence or the complete variable region sequence. Thus, one skilled in the art would know how to generate full-length antibodies according to the invention.
As used herein, the term "human antibody" is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.
As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that contains human antibody constant domains and non-human variable domains that have been modified to have a high level of sequence homology with human variable domains. This can be achieved by grafting six non-human antibody Complementarity Determining Regions (CDRs) which together form an antigen binding site onto homologous human acceptor Framework Regions (FRs) (see WO92/22653 and EP 0629240). In order to fully reconstitute the binding affinity and specificity of a parent antibody, it may be necessary to replace framework residues from the parent antibody (i.e. the non-human antibody) with human framework regions (reverse mutation). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, predominantly human framework regions, which optionally comprise one or more amino acid back mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, other amino acid modifications, not necessarily reverse mutations, may be applied to obtain a humanized antibody with preferred properties such as affinity and biochemical properties.
As used herein, the term "Fc region" refers to a region comprising at least a hinge region, a CH2 region, and a CH3 region in the direction from the N-terminus to the C-terminus of an antibody. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.
As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of the Human IgG1 antibody corresponds to amino acid 216-. However, the hinge region may also be any other subtype as described herein.
As used herein, the term "CH 1 region" or "CH 1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of the human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering as set forth in Kabat (supra). However, the CH1 region may also be of any other subtype as described herein.
As used herein, the term "CH 2 region" or "CH 2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of the human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering as set forth in Kabat (supra). However, the CH2 region may also be of any other subtype as described herein.
As used herein, the term "CH 3 region" or "CH 3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of the human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering as set forth in Kabat (supra). However, the CH3 region may also be of any other subtype as described herein.
As used herein, the term "Fc-mediated effector function" is intended to refer to a function that is the result of binding of a polypeptide or antibody to its target or antigen on a cell membrane, wherein the Fc-mediated effector function is attributable to the Fc region of the polypeptide or antibody. Examples of Fc-mediated effector functions include (i) C1q binding, (ii) complement activation, (iii) Complement Dependent Cytotoxicity (CDC), (iv) antibody dependent cell mediated cytotoxicity (ADCC), (v) Fc-gamma receptor (FcgR) binding, (vi) antibody dependent fcyr mediated antigen crosslinking, (vii) Antibody Dependent Cellular Phagocytosis (ADCP), (viii) Complement Dependent Cellular Cytotoxicity (CDCC), (ix) complement enhanced cytotoxicity, (x) binding of opsonic antibodies to complement receptors mediated by antibodies, (xi) opsonization, and a combination of any of (xi) to (xi).
As used herein, the terms "inert", "inert" or "non-activated" refer to at least the inability to bind any Fc γ R, induce Fc-mediated Fc γ R crosslinking or induce Fc γ R-mediated target antigen crosslinking via the two Fc regions of the respective antibodies, or the inability to bind the Fc region of C1 q. Monospecific or bispecific versions of antibodies can be used to test the inertness of the Fc region of an antibody.
The term "full length" when used in the context of an antibody means that the antibody is not a fragment, but contains all of the domains of that particular isotype that are normally found in nature for that isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains of the IgG1 antibody.
In the context of the present invention, the term "monovalent antibody" refers to an antibody molecule that can interact with a specific epitope on an antigen by means of only one antigen binding domain (e.g. one Fab arm). In the context of bispecific antibodies, "monovalent antibody binding" means that the bispecific antibody binds to one particular epitope on an antigen by means of only one antigen binding domain (e.g., one Fab arm).
In the context of the present invention, the term "monospecific antibody" refers to an antibody having binding specificity for only one epitope. The antibody may be a monospecific monovalent antibody (i.e., carrying only one antigen binding region) or a monospecific bivalent antibody (i.e., an antibody having two identical antigen binding regions).
The term "bispecific antibody" refers to an antibody having two different antigen binding domains, e.g., two different Fab arms or two Fab arms having different CDR regions. In the context of the present invention, bispecific antibodies are specific for at least two different epitopes. Such epitopes may be on the same or different antigens or targets. If the epitopes are on different antigens, such antigens may be on the same cell or on different cells, cell types or structures, such as extracellular matrix or vesicles and soluble proteins. Thus, bispecific antibodies may be capable of crosslinking multiple antigens, e.g., two different cells.
The term "bivalent antibody" refers to an antibody having two antigen binding regions that bind to one or two targets or epitopes on an antigen or bind to one or two epitopes on the same antigen. Thus, the bivalent antibody may be a monospecific bivalent antibody or a bispecific bivalent antibody.
The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. The amino acid is an amine (-NH)2) And a carboxyl (-COOH) functional group and a side chain (R group) specific to each amino acid. In the context of the present invention, amino acids can be classified based on their structural and chemical properties. Thus, the amino acid classes can be reflected in one or both of the following tables:
main classification based on the structural and general chemical characterization of the R group
Categories Amino acids
Acidic residue D and E
Basic residue K, R and H
Hydrophilic uncharged residues S, T, N and Q
Aliphatic uncharged residues G, A, V, L and I
Nonpolar uncharged residues C, M and P
Aromatic residue F, Y and W
Alternative physical and functional classifications of amino acid residues
Figure BDA0002720035660000221
Substitution of one amino acid for another can be classified as a conservative or non-conservative substitution. In the context of the present invention, a "conservative substitution" is the substitution of one amino acid by another amino acid having similar structural and/or chemical characteristics, such as the substitution of one amino acid residue for another amino acid residue of the same class as defined in either of the two tables above: for example, leucine can be substituted with isoleucine because they are both aliphatic, branched hydrophobes. Similarly, aspartic acid can be substituted with glutamic acid because they are small negatively charged residues.
In the context of the present invention, substitutions in an antibody are represented as:
original amino acid-position-substituted amino acid;
with reference to accepted amino acid nomenclature, a three letter code or one letter code is used, including the code "Xaa" or "X" to denote any amino acid residue. Thus, Xaa or X may generally represent any of the 20 naturally occurring amino acids. As used herein, the term "naturally occurring" refers to any one of the following amino acid residues; glycine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartic acid, asparagine, glutamic acid, glutamine, proline, tryptophan, phenylalanine, tyrosine, methionine, and cysteine. Thus, the symbol "K409R" or "Lys 409 Arg" means that the antibody comprises a substitution of lysine with arginine at amino acid position 409.
Substitution of an amino acid at a given position with any other amino acid is referred to as:
original amino acid-position; or for example "K409"
For modifications in which one or more of the original amino acids and/or one or more of the substituted amino acids may comprise more than one, but not all, amino acids, more than one amino acid may be separated by "," or "/". For example, the substitution of lysine at position 409 with arginine, alanine, or phenylalanine is:
"Lys 409Arg, Ala, Phe" or "Lys 409 Arg/Ala/Phe" or "K409R, A, F" or "K409R/A/F" or "K409 to R, A or F".
Such names may be used interchangeably in the context of the present invention, but have the same meaning and purpose.
Furthermore, the term "substitution" includes substitution to any one or other 19 natural amino acids, or to other amino acids, such as unnatural amino acids. For example, the substitution of amino acid K in position 409 includes each of the following substitutions: 409A,409C,409D,409E,409F,409G,409H,409I,409L,409M,409N,409Q,409R,409S,409T,409V,409W,409P, and 409Y. Incidentally, this is equivalent to the name 409X, where X represents any amino acid other than the original amino acid. These substitutions may also be referred to as K409A, K409C etc or K409A, C etc or K409A/C/etc. Similarly, this applies to each position mentioned herein to specifically include any of such substitutions herein.
The antibodies according to the invention may also comprise deletions of amino acid residues. Such deletions may be denoted as "del" and include, for example, the write as K409 del. Thus, in such embodiments, the lysine in position 409 has been deleted from the amino acid sequence.
As used herein, the term "host cell" is intended to refer to a cell that has received an introduction of an expression vector. It should be understood that such terms are not intended to refer to particular subject cells, but to the progeny of such cells. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK 293 cells, Expi293F cells, per.c6 cells, NS0 cells, and lymphocytes, as well as prokaryotic cells, such as e.coli, and other eukaryotic hosts, such as plant cells and fungi.
As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, e.g., CHO cells, per.c6 cells, NS0 cells, HEK 293 cells, Expi293F cells, plant cells or fungi, including yeast cells, that express the antibody or target antigen.
For The purposes of The present invention, sequence identity between two amino acid sequences is determined using The Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol.48: 443. sup. 453), as implemented in The Needle program of The EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet.16: 276. sup. 277), preferably version 5.0.0 or more. The parameters used are gap open penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:
(same residue x 100)/(alignment length-total number of gaps in alignment).
Retention of similar residues may also or alternatively be measured by similarity scores, as determined by using BLAST programs (e.g., BLAST 2.2.8 obtained via NCBI, using the standard settings BLOSUM62, open gap ═ 11 and extended gap ═ 1). Suitable variants typically exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95% or more (e.g., about 99%) similarity to the parent sequence.
As used herein, the term "internalization" or "internalization" refers to the biological process by which molecules, such as antibodies according to the invention, are phagocytosed by the cell membrane and taken up into the interior of the cell. Internalization may also be referred to as "endocytosis".
Antibodies
In a first aspect, the invention provides an antibody comprising at least one antigen binding region capable of binding to 5T4 (trophoblast glycoprotein), wherein the antibody is capable of blocking the binding to 5T4 of an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 5 and a VL region comprising the sequence shown as SEQ ID NO. 9 [059],
b) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 12 and a VL region [076] comprising the sequence shown as SEQ ID NO 16,
c) An antibody comprising a VH region comprising the sequence shown as SEQ ID NO 19 and a VL region [085] comprising the sequence shown as SEQ ID NO 23,
d) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 26 and a VL region [106] comprising the sequence shown as SEQ ID NO 30,
e) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 33 and a VL region [127] comprising the sequence shown as SEQ ID NO 37,
f) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 40 and a VL region [207] comprising the sequence shown as SEQ ID NO 44; and
g) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 47 and a VL region comprising the sequence shown as SEQ ID NO. 51 [226 ].
In particular, the invention provides an antibody comprising at least one antigen binding region capable of binding 5T4 (trophoblast glycoprotein), wherein the antibody is capable of blocking the binding of an antibody [059] comprising a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID No. 5 and a light chain Variable (VL) region comprising the sequence set forth in SEQ ID No. 9 to 5T 4.
In particular, the antibody may be capable of blocking binding to 5T4 of an antibody selected from the group consisting of:
a) An antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:40 and a light chain Variable (VL) region [207] comprising the sequence shown as SEQ ID NO:44,
b) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:47 and a light chain Variable (VL) region [226] comprising the sequence shown as SEQ ID NO: 51; and
an antibody comprising a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID NO. 5 and a light chain Variable (VL) region comprising the sequence set forth in SEQ ID NO. 9 [059 ].
In a particular embodiment of the invention, the antibody is capable of blocking binding to 5T4 of an antibody selected from the group consisting of:
a) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:40 and a light chain Variable (VL) region [207] comprising the sequence shown as SEQ ID NO: 44; and
b) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:47 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO:51 [226 ].
The antibody according to the present invention is characterized by having specificity to human (homo sapiens) 5T4 or having the ability to bind to human (homo sapiens) 5T 4. Thus, 5T4 as referred to herein may be in particular the mature polypeptide of human 5T4, as SEQ ID NO: 1.
In further embodiments, the antibodies of the invention are characterized by having specificity for cynomolgus monkey (cynomolgus monkey) 5T4 or having the ability to bind to cynomolgus monkey (cynomolgus monkey) 5T4, e.g. having specificity for both human and cynomolgus monkey 5T4 or having the ability to bind to both human and cynomolgus monkey 5T 4. Cynomolgus monkey 5T4 may in particular be the mature polypeptide of SEQ ID NO. 2.
In a further embodiment, the antibody of the invention has specificity for chicken (rooster) 5T4 or has the ability to bind to chicken (rooster) 5T4, for example, specificity for human 5T4 and chicken 5T4 or the ability to bind to human 5T4 and chicken 5T4, or specificity for human, cynomolgus monkey and chicken 5T4 or the ability to bind to human, cynomolgus monkey and chicken 5T4, wherein in particular chicken 5T4 may have the amino acid sequence of the mature polypeptide of SEQ ID No. 3.
Thus, the antibodies of the invention may have specificity for the mature polypeptide of human 5T4 as SEQ ID NO:1 and the mature polypeptide of cynomolgus monkey 5T4 as SEQ ID NO:2 or may be capable of binding to the mature polypeptide of human 5T4 as SEQ ID NO:1 and the mature polypeptide of cynomolgus monkey 5T4 as SEQ ID NO: 2.
Furthermore, the antibodies according to the invention may have specificity for human 5T4, mature polypeptide as SEQ ID NO:1, cynomolgus monkey 5T4, mature polypeptide as SEQ ID NO:2 and chicken 5T4, mature polypeptide as SEQ ID NO:3 or may be capable of binding to human 5T4, mature polypeptide as SEQ ID NO:1, cynomolgus monkey 5T4, mature polypeptide as SEQ ID NO:2 and chicken 5T4, mature polypeptide as SEQ ID NO: 3.
The antibodies according to the invention may be capable of responding to a K of 1E-7M or lessDA value, such as K, of about 1E-7M or lessDValues, 5E-8M or less, about 5E-8M or less, 1E-8M or less, about 1E-8M or less, 5E-9M or less, about 5E-9M or less, such as 1E-9M or less or such as about 1E-9M or less, binding affinity, e.g., in the range of 1E-7to 5E-10M, such as about 1E-7to about 5E-10M, such as 1E-7to 1E-9M, such as about 1E-7to about 1E-7E-9M, such as 5E-8 to 5E-10M, such as about 5E-8 to about 5E-10M, such as 5E-8 to 1E-9M, such as about 5E-8 to about 1E-9M, such as 1E-8 to 5E-10M, such as about 1E-8 to about 5E-10M, such as 1E-8 to 1E-9M, such as about 1E-8 to about 1E-9M, such as 1E-8 to 5E-9M or K in the range of about 1E-8 to about 5E-9MDThe binding affinities of values bind to human 5T4, cynomolgus monkey and/or chicken 5T 4.
Although it is within the ability of the skilled person to determine the affinity of an antibody for binding to its target, the binding affinity of an antibody according to the invention to 5T4 may in particular be determined by biolayer interferometry, optionally as set forth in example 2 herein.
More specifically, the binding affinity of an antibody according to the invention can be determined using a program, such as a biolayer interferometry program, comprising the steps of:
I) An amount of 1. mu.g/mL of the antibody was immobilized on an anti-human IgG Fc capture biosensor for 600 seconds;
II) 5T4ECDHis (mature protein of SEQ ID NO: 99) or cynomolgus monkey 5T4 (mature protein of SEQ ID NO: 2) or recombinant cynomolgus monkey 5T4 protein (Cusabio; cat number CSB-MP024093MOV) and association over a period of 200 seconds and dissociation over a period of 1000 seconds,
III) reference data against buffer control (0 nM).
In particular, the binding affinity of an antibody according to the invention, which is a monospecific bivalent antibody, such as the antibody of full-length IgG1, may be determined using an antibody as defined in any of the preceding claims.
In a further embodiment of the invention, the antibody recognizes or binds an epitope or an antibody binding region or binding site on 5T4, which binding site or epitope or antibody binding region is recognized by any one of the antibodies selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 5 and a VL region comprising the sequence shown as SEQ ID NO. 9 [059],
b) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 12 and a VL region [076] comprising the sequence shown as SEQ ID NO 16,
c) An antibody comprising a VH region comprising the sequence shown as SEQ ID NO 19 and a VL region [085] comprising the sequence shown as SEQ ID NO 23,
d) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 26 and a VL region [106] comprising the sequence shown as SEQ ID NO 30,
e) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 33 and a VL region [127] comprising the sequence shown as SEQ ID NO 37,
f) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 40 and a VL region [207] comprising the sequence shown as SEQ ID NO 44; and
g) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 47 and a VL region comprising the sequence shown as SEQ ID NO. 51 [226 ].
In a further embodiment, the antibody according to the invention recognizes or binds an antibody binding region, binding site or epitope on 5T4 which is not an antibody binding region or binding site or epitope bound by an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO:87 and a VL region comprising the sequence shown as SEQ ID NO:88 [ H8],
b) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO:83 and a VL region comprising the sequence shown as SEQ ID NO:84 [ A1 ]; and
c) An antibody comprising a VH region comprising the sequence shown in SEQ ID NO:85 and a VL region having the sequence shown in SEQ ID NO:86 [ A3 ].
In other embodiments, binding of an antibody according to the invention to 5T4 is blocked by binding of 5T4 to an antibody [ A3] comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:85 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO: 86. The antibody comprising the VH and VL sequences shown in SEQ ID NOs 85 and 86, respectively, is one of the three murine 5T4 antibodies disclosed in antibody a3, WO 2007106744. And (3) rewriting: antibody a3 with a single amino acid substitution. In the CDR sequences?
In other embodiments, antibodies according to the invention show replacement of antibodies binding to 5T4 or to the His-tagged extracellular domain of 5T4 (e.g., the mature protein of 5T4ECDHis/SEQ ID NO: 99), said antibodies binding to 5T4 comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:85 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO:86 [ A3 ]. Such displacement behavior indicates that the antibody of the invention binds an epitope that is different from the epitope bound by antibody A3, but may be adjacent to or even overlap with the epitope bound by A3.
The ability to "displace" or displace bound antibody can be determined in a biolayer interferometry assay, for example, in an assay performed as described in example 4 of the present application.
The ability of an "cross-blocking" or antibody as defined according to the invention to block binding of another antibody to 5T4 can be determined by using a Fluorescence Activated Cell Sorting (FACS) assay, for example in an assay performed as described in example 5.
In particular, the ability of an "cross-blocking" or antibody according to the invention to block the binding of another antibody to 5T4 is determined as the ability of the unconjugated antibody to block the conjugated antibody, and optionally in a procedure comprising the following steps:
i) providing a set of samples, each sample comprising a mixture of human ovarian adenocarcinoma SK-OV-3 cells, an antibody that binds 5T4 and is conjugated to Fluorescein Isothiocyanate (FITC), and an excess of unconjugated antibody that targets 5T4,
ii) incubating the sample at 4 ℃ for 30 minutes and then centrifuging the sample,
iii) removing the supernatant from each sample and resuspending the cells in buffer and determining the Mean Fluorescence Intensity (MFI) of FITC using a flow cytometer; and is
iv) percent binding was calculated as follows:
The difference in MFI between cells incubated with the mixture of FITC-conjugated and unconjugated antibodies and cells not incubated with FITC-conjugated or unconjugated antibodies was multiplied by 100 and then divided by the difference in MFI between cells incubated with the mixture of FITC-conjugated and IgG-b12 antibodies and cells not incubated with FITC-conjugated or unconjugated antibodies.
Although the skilled person will be familiar with suitable techniques for determining the ability of an antibody to block the binding of another antibody to its target or displace another antibody bound to its target, the present application discloses procedures suitable for determining blocking of binding and displacement. Thus, in some embodiments, biolayer interferometry may be used to determine the ability of an antibody according to the invention to block the binding of another antibody to 5T4 or displace another antibody that binds to 5T4, for example in a biolayer interferometry performed as described in example 3.
In particular, biolayer interferometry may be used to determine the ability of an antibody according to the invention to block the binding of another antibody to 5T4 or displace another antibody that binds to 5T4, which may be determined in a procedure comprising the following steps:
i) The antibody according to the present invention was immobilized to the activated amine-reactive second-generation biosensor in an amount of 20. mu.g/mL in 10mM sodium acetate buffer,
ii) quenching the biosensor with immobilized antibody in ethanolamine at pH 8.5,
iii) the biosensor with immobilized antibody was immersed in a composition comprising 3.6. mu.g/mL (100nM) of human 5T4ECDHI (mature protein of SEQ ID NO: 99) for a period of 500 seconds, followed by
iv) immersing the biosensor with immobilized antibody and 5T4ECDHis in a composition comprising 10 μ g/mL of an additional antibody targeting 5T4 and determining the association response over a period of 500 seconds;
wherein steps i) -iv) are performed at a temperature of 30 ℃ and with shaking at 1000 rpm.
The antibodies provided herein can bind to an epitope or antibody binding region on human 5T4 comprising amino acid residues R73, Y92, and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Also provided herein are antibodies that bind to an epitope or antibody binding region on human 5T4 comprising amino acid residues S69, R73, Y92, and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Also provided herein are antibodies that bind to an epitope or antibody binding region on human 5T4 comprising amino acid residues R73, T74, Y92, R94, and N95; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Based on the results provided in example 16 herein, it is hypothesized, without wishing to be bound by theory, that any one or more of these amino acid residues (i.e., S69, R73, T74, Y92, R94, and N95) are directly involved in antibody binding, e.g., through non-covalent interactions; for example, amino acid residues within the CDR sequences of an antibody. This hypothesis is supported by the fact that these residues were identified as surface exposed on the structure of 5T4 (4 CNM; supplied by the RCSB PDB protein database; DOI:10.2210/PDB4 CNM/PDB); such as those disclosed in Zhao, Y., Malinauskas, T., Harlos, K., & Jones, E.Y. (2014.) Structural insulators into the inhibition of Wnt signaling by cancer inhibitor 5T4/Wnt-activated inhibition factor 1, Structure,22(4), 612-.
One or more of the following additional amino acid residues may be involved in binding of the antibody, e.g. indirectly involved in binding, e.g. by influencing the folding of the protein and/or the positioning of one or more amino acid residues directly involved in antibody binding: l89, F111, L117, F138, L144, D148, N152; the numbering of each amino acid residue refers to its position in SEQ ID NO 1. In particular, L89, F111, L117, F138, L144 have been identified as part of the hydrophobic core within 5T4 as described in Zhao et al, Structure,22(4), 612-.
Furthermore, the antibodies disclosed herein may be directed to an epitope on human 5T4 or an antibody binding region within which amino acid residues R73, Y92 and R94 are directly involved in binding the antibody, and wherein one or more of amino acid residues F111, F138, L144 and D148 are indirectly involved in said binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
The antibodies provided herein can bind to an epitope on human 5T4 or an antibody binding region within which amino acid residues S69, R73, Y92 and R94 are directly involved in binding to the antibody, and wherein one or more of amino acid residues F111, F138 and D148 are indirectly involved in the binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Furthermore, the present disclosure provides antibodies that bind to an epitope on human 5T4 or an antibody binding region within which amino acid residues R73, T74, Y92, R94 and N95 are directly involved in binding to the antibody, and wherein amino acid residue F138 is indirectly involved in the binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
The amino acid residues comprised by the epitope or antibody binding region and optionally one or more further amino acid residues indirectly involved in binding may be identified by alanine scanning of human 5T4 having the amino acid sequence shown in SEQ ID NO:1 or the mature polypeptide sequence of SEQ ID NO:1 or by alanine scanning of a polypeptide comprising amino acid residues 32-355 of SEQ ID NO: 1.
The alanine scan can be specifically as described in example 16 herein or substantially as described in example 16 herein.
In addition, alanine scanning can be performed by a procedure comprising the following steps:
i) expressing a mutant human 5T4 polypeptide and a wild type 5T4 polypeptide (amino acid residues 32-355 of SEQ ID NO: 1) individually in human embryonic kidney cells, such as HEK 293 cells, to provide a sample comprising 70-90.000 cells, such as 80.000 cells, for each mutant or wild type 5T4, in which mutant human 5T4 polypeptide all amino acid residues in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO: 1) are individually substituted with alanine, except cysteine and alanine,
ii) incubating the cells in each sample with 20 μ L of the antibody conjugated with Fluorescein Isothiocyanate (FITC) antibody (3 μ g/mL; in FACS buffer) and then in 150-; lonza, catalog number BE17-517]+0.1％[w/v]BSA [ Roche, Cat No. 10735086001]+0.02％[w/v]Sodium azide [ NaN3(ii) a EMELCA Bioscience, catalog number 41920044-3]) Washing each sample twice and resuspending the cells in each sample in 30. mu.L of FACS buffer,
iii) for each sample, determining the average amount of antibody bound per cell as the geometric mean of fluorescence intensity (gMFI) of the population of living single cells in the sample, and normalizing the data for each test antibody against the binding intensity of a non-cross-blocking 5T 4-specific control antibody using the following equation:
Figure BDA0002720035660000311
wherein "aa position" refers to a position mutated to alanine,
wherein a Z-score is calculated to express the loss or gain of antibody binding according to the following calculation:
Figure BDA0002720035660000312
where μ and σ are the mean and standard deviation, respectively, of the normalized gMFI calculated from all mutants,
wherein the gMFI of the control antibody is lower than the average gMFI for a particular 5T4 mutantControl antibody2.5 Xaverage gMFIControl antibodySD (from all mutants) of (c), data were excluded from the analysis; and optionally
Wherein data is excluded from the analysis if a residue binds with a Z-score just below-1.5 (e.g., -1.5 to-1.8, such as-1.5 to-1.7 or such as-1.5 to-1.6) and the residue is predicted to be buried and spatially separated from the majority of residues that are predicted to be surface exposed and for which loss of or reduced binding is determined.
Suitable non-cross-blocking 5T 4-specific control antibodies to be used in step iii) are bispecific antibodies comprising
-an antigen binding region comprising the VH sequence shown as SEQ ID NO:83 and the VL sequence shown as SEQ ID NO:84 [ a1 ]; and
an antigen-binding region comprising the VH sequence shown as SEQ ID NO:97 and the VL sequence shown as SEQ ID NO:98 [ B12 ].
The invention provides antibodies that bind 5T4 such that if any one or more of amino acid residues R73, Y92 and R94 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
In particular, if any one or more of amino acid residues S69, R73, Y92 and R94 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Furthermore, if any one or more of amino acid residues R73, T74, Y92, R94, and N95 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Likewise, the antibodies disclosed herein can bind 5T4 such that if any one or more of amino acid residues L89, F111, L117, F138, L144, D148, N152 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
Furthermore, the antibody may bind 5T4 such that if any one or more of amino acid residues R73, Y92, R94, F111, F138, L144 and D148 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
The antibody may bind 5T4 such that if any one or more of amino acid residues S69, R73, Y92, R94, F111, F138 and D148 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
In other embodiments, the antibodies of the invention may bind 5T4 such that if any one or more of amino acid residues R73, T74, Y92, R94, N95, and F138 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
The effect of any alanine substitution provided above can be determined by alanine scanning a polypeptide comprising amino acid residues 32-355 of SEQ ID NO: 1.
In particular, the effect of alanine substitution can be determined by the procedure described in example 16 herein or substantially as described in example 16 herein.
Loss of binding may be defined as a Z score of less than 1.5 for binding; the Z-score is optionally calculated as described in example 16 herein or substantially as described in example 16 herein.
The effect of alanine substitution can be determined by a procedure comprising the following steps:
i) expressing a mutant human 5T4 polypeptide and a wild-type 5T4 polypeptide individually in human embryonic kidney cells, such as HEK 293 cells, to provide a sample comprising 70-90.000 cells, such as 80.000 cells, for each mutant or wild-type 5T4, in which mutant human 5T4 polypeptide all amino acid residues in the human 5T4 extracellular domain (corresponding to amino acid residues 32-355 of SEQ ID NO: 1) are individually substituted with alanine, except cysteine and alanine,
ii) incubating the cells in each sample with 20 μ L of the antibody conjugated with Fluorescein Isothiocyanate (FITC) antibody (3 μ g/mL; in FACS buffer) and then in 150-; lonza, catalog number BE17-517]+0.1％[w/v]BSA [ Roche, Cat No. 10735086001]+0.02％[w/v]Sodium azide [ NaN3(ii) a EMELCA Bioscience, catalog number 41920044-3]) Washing each sample twice and resuspending the cells in each sample in 30. mu.L of FACS buffer,
iii) for each sample, determining the average amount of antibody bound per cell as the geometric mean of fluorescence intensity (gMFI) of the population of living single cells in the sample, and normalizing the data for each test antibody against the binding intensity of a non-cross-blocking 5T 4-specific control antibody using the following equation:
Figure BDA0002720035660000331
wherein "aa position" refers to a position mutated to alanine,
wherein a Z-score is calculated to express the loss or gain of antibody binding according to the following calculation:
Figure BDA0002720035660000332
where μ and σ are the mean and standard deviation, respectively, of the normalized gMFI calculated from all mutants,
wherein the gMFI of the control antibody is lower than the average gMFI for a particular 5T4 mutantControl antibody2.5 Xaverage gMFIControl antibodySD (from all mutants) of (c), data were excluded from the analysis; and optionally
Wherein data is excluded from the analysis if a residue binds with a Z-score just below-1.5 (e.g., -1.5 to-1.8, such as-1.5 to-1.7 or such as-1.5 to-1.6) and the residue is predicted to be buried and spatially separated from the majority of residues that are predicted to be surface exposed and for which loss of or reduced binding is determined.
Suitable non-cross blocking 5T 4-specific control antibodies in step iii) of the foregoing procedure are bispecific antibodies comprising
-an antigen binding region comprising the VH sequence shown as SEQ ID NO:83 and the VL sequence shown as SEQ ID NO:84 [ a1 ]; and
an antigen-binding region comprising the VH sequence shown as SEQ ID NO:97 and the VL sequence shown as SEQ ID NO:98 [ B12 ].
The antibodies according to the invention may be characterized as having reduced internalization ability as shown by reduced cytotoxicity when conjugated to a cytotoxic moiety as compared to an identically conjugated antibody [ H8] containing a heavy chain Variable (VH) region comprising the sequence shown in SEQ ID No. 87 and a light chain Variable (VL) region comprising the sequence shown in SEQ ID No. 88. The antibody comprising the VH and VL sequences shown in SEQ ID NOS: 87 and 88, respectively, can be the murine 5T4 antibody mAb5T4, also known as the H8 antibody (Shaw et al (2002), biochem. J.363:137-45, WO 98/55607). Various chimeric or humanized forms of antibody H8 are disclosed in WO 06/031653.
The cytotoxicity or internalization of a 5T4 antibody that binds monovalent to 5T4 can be determined using the procedure described in example 7 herein. In particular, cytotoxicity may be determined in an assay comprising the following steps:
i) There is provided a toxin-conjugated bispecific antibody monovalent binding to 5T4 comprising a first Fab arm of an antibody as defined in any one of the preceding claims and a second Fab arm capable of binding to the HIV viral protein gp120(HIV-1gp120), wherein the HIV-1gp 120-specific Fab arm is conjugated to Duostatin-3,
ii) 5T4 positive breast cancer cells MDA-MB-468(ATCC clone HTB-132) or HCC1954(ATCC clone CRL-1338) were incubated with the bispecific antibody that binds monovalent to 5T4 for 5 days at 37 ℃; and
iii) determining the viability of the cells.
IgG-b12 is an HIV-1gp 120-specific antibody (Barbas, CF. J Mol biol.1993Apr 5; 230(3): 812-23). The sequences of the heavy (VH) and light chain Variable (VL) regions are set forth in SEQ ID NOS: 97 and 98, respectively.
In certain embodiments, the antibodies of the invention are antibodies, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 6, 7 and 8 [059],
b) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 13, 14 and 15 [076],
c) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [085] of SEQ ID Nos. 20, 21 and 22,
d) A heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [106] of SEQ ID Nos. 27, 28 and 29,
e) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [127] of SEQ ID NOS: 34, 35 and 36,
f) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [207] of SEQ ID Nos. 41, 42 and 43,
g) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [226] of SEQ ID Nos. 48, 49 and 50, and
h) a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3 sequences collectively comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to g).
In other embodiments, the antibody according to the invention is an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 6, 7 and 8 [059],
b) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [207] of SEQ ID Nos. 41, 42 and 43,
c) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID nos. 48, 49 and 50 [226 ]; and
d) A heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3 sequences collectively comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to c).
In particular, the antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8 [059 ].
Alternatively, the antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of: a heavy chain Variable (VH) region [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 41, 42 and 43.
The antibody according to the invention may also be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of: a heavy chain Variable (VH) region [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 48, 49 and 50.
In other embodiments, the antibody according to the invention is an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region selected from the group consisting of:
a) A heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 6, 7 and 8, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 10, AAS and SEQ ID NO 11, respectively [059],
b) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 13, 14 and 15, respectively, and a light chain variable region (VL) [076] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 17, DAS and SEQ ID NO:18, respectively,
c) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 20, 21 and 22, respectively, and a light chain variable region (VL) [085] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 24, DAS and SEQ ID NOs 25, respectively,
d) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 27, 28 and 29, respectively, and a light chain variable region (VL) [106] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:31, DVS and SEQ ID NO:32, respectively,
e) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 34, 35 and 36, respectively, and a light chain variable region (VL) [127] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:38, DAS and SEQ ID NO:39, respectively,
f) a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 41, 42 and 43, respectively, and a light chain variable region (VL) [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, DAS and SEQ ID NO:46, respectively,
g) A heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 48, 49 and 50, respectively, and a light chain variable region (VL) [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:52, DAS and SEQ ID NO:53, respectively; and
h) a heavy chain variable region (VH) and a light chain variable region (VL), each region comprising a CDR1, CDR2 and CDR3 sequence, said CDR1, CDR2 and CDR3 sequences collectively comprising at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or at most 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to g).
The antibody according to the invention may be an antibody, wherein the six Complementarity Determining Regions (CDRs) capable of binding the antigen binding region of 5T4 collectively comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or at most 10 amino acid substitutions when compared to;
iv) the CDR sequences of SEQ ID NO 6, 7, 8, 10, AAS and SEQ ID NO 11 [059],
v) CDR sequences [207] of SEQ ID NO: 41, 42, 43, 45, DAS and SEQ ID NO: 46; or
vi) CDR sequences of SEQ ID NO 48, 49, 50, 52, DAS and SEQ ID NO 53 [226 ].
Preferably, 1 of the amino acid substitutions, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, are conservative amino acid substitutions.
The antibody may in particular comprise one or two heavy chain variable regions, wherein the complementarity determining region 3(CDR3) comprises six consecutive amino acid residues [059, 207, 226] of the sequence shown in SEQ ID NO:102 (YYGMDV). The six consecutive amino acid residues may be the C-most amino acid residue within CDR 3.
The antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises one or two heavy chain variable region (VH) comprising the CDR1 sequence of SEQ ID NO:41(GGSFSGYY), SEQ ID NO:103 (IDHSX)1ST) and the CDR2 sequence of SEQ ID NO 104 (AX)2WFGELX3X4YYYGMDV) and the light chain variable region comprises the CDR3 sequence of SEQ ID NO 105 (QSVSSX)5) The CDR1 sequence, the CDR2 sequence DAS and the CDR3 sequence of SEQ ID NO:46(QQRSNWPLT) of (I), wherein X1Is G or E, X2Is A or G, X3Is W or Y, X4Is D or H, and X5Is Y or F [207, 226]。
The antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8 respectively and a light chain variable region (VL) [059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 10, AAS and 11 respectively.
Alternatively, the antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID nos. 41, 42 and 43, respectively, and a light chain Variable (VL) region [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 45, DAS and SEQ ID NO 46, respectively.
Additionally, the antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 sequences of SEQ ID No. 48, 49 and 50, respectively, and a light chain variable region (VL) [226] comprising CDR1, CDR2 and CDR3 sequences of SEQ ID No. 52, DAS and SEQ ID No. 53, respectively.
In some embodiments, the antibody according to the invention is an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) selected from the group consisting of:
a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or a sequence [059] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:5,
b) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:12 [076],
c) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:19 or a sequence [085] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:19,
d) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:26 or a sequence [106] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:26,
e) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:33 or a sequence [127] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:33,
f) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence [207] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 40; and
g) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 [226 ].
The antibody according to the invention may in particular be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID No. 5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID No. 5 [059 ].
Likewise, an antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:40 [207 ].
In addition, the antibody according to the invention may be an antibody, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 [226 ].
In other embodiments, the antibody according to the invention is an antibody, wherein said antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:
a) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO. 5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 5 and a light chain variable region (VL) [059] comprising the sequence of SEQ ID NO. 9 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 9,
b) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO 12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 12 and a light chain variable region (VL) [076] comprising the sequence of SEQ ID NO 16 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 16,
c) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO 19 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 19 and a light chain variable region (VL) [085] comprising the sequence of SEQ ID NO 23 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 23,
d) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO 26 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 26 and a light chain variable region (VL) [106] comprising the sequence of SEQ ID NO 30 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 30,
e) A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:33 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:33 and a light chain variable region (VL) 127 comprising the sequence of SEQ ID NO:37 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:37,
f) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:40 and a light chain variable region (VL) 207 comprising the sequence of SEQ ID NO:44 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:44,
g) a heavy chain variable region (VH) comprising the sequence of SEQ ID NO 47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 47 and a light chain variable region (VL) 226 comprising the sequence of SEQ ID NO 51 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 51.
In one embodiment, the at least one binding region comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region having up to 10 mutations or substitutions, up to 5 mutations or substitutions, such as up to 4 mutations or substitutions, such as up to 3 mutations or substitutions, such as up to 2 mutations or substitutions, such as up to 1 mutation or substitution, across said heavy chain variable region (VH) and light chain variable region (VL) selected from the group consisting of:
a) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO. 5 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO. 9 [059],
b) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 12 and a light chain variable region (VL) [076] comprising or consisting of the sequence of SEQ ID NO 16,
c) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 19 and a light chain variable region (VL) [085] comprising or consisting of the sequence of SEQ ID NO 23,
d) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 26 and a light chain variable region (VL) [106] comprising or consisting of the sequence of SEQ ID NO 30,
e) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 33 and a light chain variable region (VL) 127 comprising or consisting of the sequence of SEQ ID NO 37,
f) A heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 40 and a light chain variable region (VL) 207 comprising or consisting of the sequence of SEQ ID NO 44; and
g) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:47 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:51 [226 ].
In some embodiments of the present disclosure, up to 10 mutations or substitutions, up to 5 mutations or substitutions, such as up to 4 mutations or substitutions, such as up to 3 mutations or substitutions, such as up to 2 mutations or substitutions, such as 1 mutation or substitution, are allowed across the full length of the variable heavy chain and the entire variable light chain. In other embodiments, up to 10 mutations or substitutions, up to 5 mutations or substitutions, such as up to 4 mutations or substitutions, such as up to 3 mutations or substitutions, such as up to 2 mutations or substitutions, such as up to 1 mutation or substitution may not be within any of the 6 CDR sequences in the variable heavy and variable light chains.
Up to 10 mutations or substitutions may be distributed over the entire length of the variable heavy and variable light chains of each binding region. Some or all of the mutations or substitutions may be conservative substitutions, in which one amino acid residue is substituted by an amino acid residue of the same class as indicated under the definition "amino acid" above; for example, one acidic amino acid residue may be substituted for another acidic amino acid residue, and an aromatic residue may be substituted for another aromatic residue. It may be preferred that 35% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 92% or more, 93% or more or 94% or more substitutions in the variant are conservative amino acid residue substitutions.
In particular, some or all of the mutations or substitutions may be made with one or more amino acid residues, each having the same physical or functional properties as the corresponding amino acid residue it replaces. Amino acid residues sharing physical and functional properties are provided under the definition "amino acid" above; for example, above under the definition "amino acid"; for example, a hydrophobic residue can be substituted for another hydrophobic amino acid residue, or a cycloalkenyl-related residue can be substituted for another cycloalkenyl-related residue.
In particular, an antibody comprising a substitution or mutation as disclosed above may be a functional variant of a VL region, a VH region or one or more CDRs as defined above with reference to a sequence identifier. Functional variants of VL, VH or CDRs for use in the context of the antibodies of the invention still allow the antibodies to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the affinity and/or specificity/selectivity of the parent antibody, and in some cases such 5T4 antibodies may even be associated with higher affinity, selectivity and/or specificity than the parent antibody.
In other embodiments of the invention, the antibody is an antibody wherein the antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from the group consisting of:
a) A heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO. 5 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO. 9 [059],
b) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 12 and a light chain variable region (VL) [076] comprising or consisting of the sequence of SEQ ID NO 16,
c) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 19 and a light chain variable region (VL) [085] comprising or consisting of the sequence of SEQ ID NO 23,
d) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 26 and a light chain variable region (VL) [106] comprising or consisting of the sequence of SEQ ID NO 30,
e) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 33 and a light chain variable region (VL) 127 comprising or consisting of the sequence of SEQ ID NO 37,
f) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO 40 and a light chain variable region (VL) 207 comprising or consisting of the sequence of SEQ ID NO 44; and
g) a heavy chain variable region (VH) comprising or consisting of the sequence of SEQ ID NO:47 and a light chain variable region (VL) comprising or consisting of the sequence of SEQ ID NO:51 [226 ].
The antibody of the invention may be a full length antibody, for example a full length IgG1 antibody.
Furthermore, the antibody of the present invention may be a monovalent antibody. Alternatively, the antibody according to the invention may be a bivalent antibody.
In other embodiments, the antibodies provided according to the invention are monospecific antibodies.
Alternatively, the antibody according to the invention may be a bispecific antibody.
It is also within the scope of the present disclosure to provide an antibody as defined above comprising binding to CD3, such as human CD3(epsilon), such as human CD3(epsilon) as specified in SEQ ID NO: 4. The antigen binding region of an antibody of (1).
In particular, the present disclosure provides bispecific antibodies comprising a first antigen-binding region of an antibody as disclosed above and a second binding region that binds CD3, such as human CD3, as defined above.
Examples of bispecific antibody molecules that can be used in the present invention include, but are not limited to, (i) a single antibody having two arms comprising different antigen binding regions, (ii) a single chain antibody specific for two different epitopes, e.g., via two scfvs linked in series by an additional peptide linker; (iii) dual variable domain antibodies (DVD-Ig)TM) Wherein each light and heavy chain contains two Variable domains connected in series via a short peptide linkage, Wu et al, Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig) TM) Molecule, in Antibody Engineering, Springer Berlin Heidelberg (2010); (iv) a chemically linked bispecific (Fab') 2 fragment; (v)
Figure BDA0002720035660000421
it is a fusion of two single chain diabodies (diabodies), resulting in a tetravalent bispecific antibody, which has two binding sites for each target antigen; (vi) elastomers (flexosomes), which are a combination of scFv and diabody, resulting in multivalent molecules; (vii) so-called "docking and latching" molecules based on the "dimerization and docking domains" in protein kinase A
Figure BDA0002720035660000422
The dimerization and docking domains can produce trivalent bispecific binding proteins when applied to Fab(ii) white, consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules comprising, for example, two scfvs fused to the two ends of a human Fab arm; and (ix) diabodies.
In one embodiment, the bispecific antibody of the invention is a diabody, a cross-body such as CrossMabs, or a bispecific antibody obtained by controlled Fab arm exchange (e.g. as described in WO 2011/131746).
Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) a recombinant IgG-like dual targeting molecule, wherein the molecule is flanked on each side by a Fab fragment or a portion of a Fab fragment of at least two different antibodies; (iii) an IgG fusion molecule in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; (iv) an Fc fusion molecule in which a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, an Fc region, or portion thereof; (v) a Fab fusion molecule in which different Fab fragments are fused together, fused to a heavy chain constant domain, Fc region or portion thereof; (vi) ScFv-based and diabody-based heavy chain antibodies (e.g., domain antibodies,
Figure BDA0002720035660000434
) Wherein different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies,
Figure BDA0002720035660000431
) Fused to each other or to another protein or carrier molecule fused to a constant heavy domain, Fc region or portion thereof.
Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to
Figure BDA0002720035660000432
(Trion Pharma/Freenius Biotech, WO/2002/020039), protrusion-in-Holes (Knobs-intro-Holes) (Genentech, WO 9850431;), CrossMAbs (Roche, WO2011117329) and electrostatically matched (Amgen, EP1870459 and WO 2009089004; Chugai, US 201000155133; oncomed, WO2010129304), LUZ-Y (Genencoch), DIG-body and PIG-body (Pharmabcine), chain Exchange Engineered Domain antibodies (Strand Exchange Engineered Domain body) (SEEDbody) (EMD Serono, WO2007110205), bicronics (Merus), Fc Δ Adp (Regeneron, WO 2010/015792), bispecific IgG1 and IgG2(Pfizer/Rinat, WO11143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), bispecific antibodies (Roche WO 2009/080254) and PIG-body (Pharmabcine)
Figure BDA0002720035660000433
Molecule (Genmab A/S, WO 2011/131746).
Examples of recombinant IgG class dual targeting molecules include, but are not limited to, Dual Targeting (DT) -Ig (GSK/Domantis), two-in-one antibody (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2(F-Star, WO2008003116), Zybodes TM(Zyngenia), methods using a common light chain (Crucell/Merus, US7,262,028), kappa lambda Bodies (NovImmune) and CovX-body (CovX/Pfizer).
Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD) -IgTM(Abbott, US7,612,181), dual domain doubleheaded antibody (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecific (Imclone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec, US007951918), scFv fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc, CN 102250246) and TvAb (Roche, WO2012025525, WO 2012025530).
Examples of Fc fusion molecules include, but are not limited to, ScFv/Fc fusion (Academic institute), SCORPION (empirical BioSolutions/traunion, Zymogenetics/BMS), Dual Affinity targeting Technology (Fc-DART)TM) (MacroGenics, WO2008157379, WO2010/080538) and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine-China).
Examples of Fab fusion bispecific antibodies include, but are not limited to, F (ab)2 (Metarex/AMGEN), Dual-Action or Bis-Fab (Genentech),
Figure BDA0002720035660000441
(DNL) (ImmunoMedics), bivalent bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
Examples of scFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, bispecific T cell binding agents (engage)
Figure BDA0002720035660000442
(Micromet,Tandem Diabody(TandabTM) (Affinized), double retargeting technology (DART) (MacroGenics), single chain diabody (Academic), TCR-like antibody (AIT, Receptor Logics), human serum albumin ScFv fusion (Merrilack) and COMBODY (Epigen Biotech), double targeting nanobody
Figure BDA0002720035660000443
(Ablynx), a double-targeted heavy chain-only domain antibody.
The antibody according to the present disclosure may in particular be an antibody wherein the antigen binding region that binds CD3 comprises
A heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID nos. 54, 55, and 56, respectively; [ huCD3-H1L1] (WO2015001085(Genmab A/S));
and optionally
Light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 58, GTN and 59, respectively [ huCD3-H1L1 ].
Also disclosed are antibodies wherein the antigen binding region that binds CD3 comprises
A heavy chain variable region (VH) comprising the sequence of SEQ ID NO:57 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:57 [ huCD3-H1L1 ];
and optionally
A light chain variable region (VL) comprising the sequence of SEQ ID NO:60 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:60 [ huCD3-H1L1 ].
The present disclosure further provides antibodies, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 6, 7 and 8 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 10, AAS and SEQ ID NO 11 respectively [059],
and
the antigen binding region capable of binding CD3 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown as SEQ ID NOs 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown as SEQ ID NO:58, sequence GTN and SEQ ID NO:59, respectively [ huCD3-H1L1 ].
In addition, the present disclosure provides antibodies, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 sequences as SEQ ID No. 41, 42 and 43 and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 sequences as SEQ ID No. 45, DAS and 46 respectively [207 ];
and
the antigen binding region capable of binding CD3 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs 54, 55 and 56, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, sequence GTN and SEQ ID NO:59, respectively [ huCD3-H1L1 ].
In addition, the disclosure provides antibodies, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 52, DAS and SEQ ID NOs 53 [226-VH + VL CDR1, -2 and-3 sequences ];
and
the antigen binding region capable of binding CD3 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOS: 54, 55 and 56, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NO:58, CDR GTN and SEQ ID NO:59, respectively [ huCD3-H1L1 ].
An antigen-binding region that binds CD3 may have an equilibrium dissociation constant K in the range of 200-1000 nM, such as in the range of 300-1000 nM, in the range of 400-1000 nM, in the range of 500-1000 nM, in the range of 300-900 nM, in the range of 400-700 nM, in the range of 500-900 nM, in the range of 500-800 nM, in the range of 500-700 nM, in the range of 600-1000 nM, in the range of 600-900 nM, in the range of 600-800 nM, or such as in the range of 600-700 nM DAnd (4) combining.
In a further embodiment, the antibodies disclosed herein have a lower binding affinity for human CD3 than an antibody having an antigen-binding region comprising a VH sequence shown as SEQ ID NO:57 and a VL sequence shown as SEQ ID NO:60 [ huCD3-H1L1], preferably wherein the affinity is at least 2-fold lower, e.g., at least 5-fold lower, such as at least 10-fold lower, e.g., at least 20-fold lower, at least 30-fold lower, at least 40-fold lower, at least 45-fold lower, at least 50-fold lower, at least 55-fold lower, or such as at least 50-fold lower.
In particular, an antigen-binding region that binds CD3 may have an equilibrium constant K within the range of 1-100 nM, such as within the range of 5-100 nM, within the range of 10-100 nM, within the range of 1-80 nM, within the range of 1-60 nM, within the range of 1-40 nM, within the range of 1-20 nM, within the range of 5-80 nM, within the range of 5-60 nM, within the range of 5-40 nM, within the range of 5-20 nM, within the range of 10-80 nM, within the range of 10-60 nM, within the range of 10-40 nM, or such as within the range of 10-20 nMDAnd (4) combining.
The affinity of an antibody according to the invention for binding to CD3 can be determined by biolayer interferometry, using a modification of the above procedure or as described in example 2 herein, wherein the antibody is immobilized on a human IgG Fc capture biosensor and the association and dissociation of CD3E27-GSKa (mature protein of SEQ ID NO: 101) with the immobilized antibody is determined. Furthermore, the affinity of an antibody according to the invention for binding to CD3 can be determined by biolayer interferometry as provided in example 9 herein.
Antibodies binding to CD3, in particular human CD3, with reduced affinity are provided in WO 2017/009442, and it will be appreciated that any of these antibodies may serve as a basis for the production of antibodies according to the invention which, in addition to the ability to bind 5T4, also have the ability to bind CD3 with reduced affinity. Thus, in other embodiments, the antibody according to the invention is an antibody wherein
The antigen binding region that binds CD3 comprises a heavy chain Variable (VH) region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence,
when compared to a heavy chain Variable (VH) region comprising the sequence shown in SEQ ID NO:57, the heavy chain Variable (VH) region has an amino acid substitution in one of the CDR sequences at a position selected from the group consisting of: t31, N57, H101, G105, S110 and Y114, the positions being numbered according to the sequence of SEQ ID NO: 57; and
the wild-type light chain Variable (VL) region comprises the CDR1, CDR2 and CDR3 sequences shown as SEQ ID NOS 58, GTN and SEQ ID NO 59, respectively.
Preferably, the CDR1, CDR2 and CDR3 of the heavy chain Variable (VH) region of the antigen binding region that binds CD3 collectively comprise up to 1, 2, 3, 4 or 5 amino acid substitutions when compared to the sequence set forth in SEQ ID NO: 57.
The amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain Variable (VH) region that binds the antigen-binding region of CD3 and the amino acid sequences of the CDR1, CDR2, and CDR3 of the wild-type heavy chain Variable (VH) region can have at least 95% sequence identity, such as at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, calculated based on aligning the amino acid sequence consisting of the sequences of the CDR1, CDR2, and CDR3 of the heavy chain Variable (VH) region that binds the antigen-binding region of CD3 with the amino acid sequences comprising the CDR1, CDR2, and CDR3 sequences of the wild-type heavy chain Variable (VH) region.
In particular, the antigen binding region that binds CD3 may comprise a mutation selected from the group consisting of: T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, Y114V, the positions being numbered according to the reference sequence of SEQ ID NO: 57.
In certain embodiments, an antibody according to the invention is an antibody, wherein when the antibody is a bispecific antibody that lacks Fc-mediated effector function or has reduced Fc-mediated effector function (an "inert" antibody), and comprises the antigen binding region of an antibody that binds CD3, then the antibody:
a) When purified Peripheral Blood Mononuclear Cells (PBMC) or T cells are used as effector cells, it is possible to mediate concentration-dependent cytotoxicity of SK-OV-3 cells, e.g., when assayed as described in example 14 herein,
b) when purified T cells are used as effector cells, it is possible to mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, e.g., when assayed as described in example 13 herein,
c) capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells; for example as determined as described in example 13(II) herein,
d) capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells; for example when measured as described in example 17 herein,
e) when purified T cells are used as effector cells, capable of inducing cytotoxicity of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells, e.g., when assayed as described in example 17 herein; and/or
f) Reconstitution of mouse xenograft models in humanized immune hematopoietic stem cells, such as NOD. Cg-Prkdc inoculated with human MDA-MB-231 tumor cellsscidIl2rgtm1WjlAnti-tumor activity, such as delayed tumor outgrowth, is shown in/SzJ; for example when measured as described in example 15; and
Furthermore, the antibody according to the invention is an antibody which does not bind leukocyte Fc γ R when assessed by flow cytometry or ELISA and does not induce CD3 antibody-dependent Fc γ R-mediated CD3 cross-linking by binding C1q in the absence of target (5T4) -specific tumor cells.
A more detailed disclosure of antibodies with reduced or no Fc-mediated effector function ("inert" antibodies) can be found below.
Determining the ability of an antibody to mediate concentration-dependent cellular cytotoxicity of SK-OV-3 cells in an in vitro cytotoxicity assay, said assay comprising the steps of:
i) isolating PBMCs or T cells from a healthy human donor buffy coat,
ii) providing
A first set of samples, wherein each sample comprises PBMCs and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of PBMCs to SK-OV-3 cells in the sample is 1:2,1:1,2:1,4:1,8:1, and 12: 1; and
a second set of samples, wherein each sample comprises T cells and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of T cells to SK-OV-3 cells in the sample is 1:2,1:1,2:1,4:1, and 8:1
iii) adding the antibody to each set of samples at a concentration ranging from 0.0128ng/mL to 1000ng/mL and incubating the samples at 37 ℃ for 72 hours; then the
iv) viability of the SK-OV-3 cells was assessed using Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide).
The ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells can be determined in an assay comprising the following steps:
i) isolating T cells from a healthy human donor buffy coat,
ii) providing a set of samples, wherein each sample comprises T cells and human breast cancer MDA-MB-231 cells, and wherein the ratio of T cells to MDA-MB-231 cells in the sample is 8:1,
iii) adding said antibody to the set of samples at a concentration ranging from 0.0128ng/mL to 1000ng/mL and incubating said samples at 37 ℃ for 72 hours,
iv) staining the T cells with fluorescently labeled antibodies against T cell activation markers, such as CD69-APC, CD25-PE-Cy7 and CD279/PD1-BV604 antibodies, by incubation with the antibodies at 4 ℃ for 30 minutes; and is
v) analyzing the sample by flow cytometry.
APC anti-human CD69(CD69-APC) antibodies are available, for example, from BioLegend (catalog nos. 310909 and 310910). The CD25 monoclonal antibody PE-Cyanine7(CD25-PE-Cy7) is also available from, for example, ThermoFisher Scientific (Cat. No. 25-0259-42) and BD Biosciences (Cat. No. 557741). Finally, the CD279/PD1-BV604 antibody was purchased from Genscript (Cat. No. A01828).
T cells can be activated in vitro in the presence of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells as determined by a procedure comprising the following steps:
i) providing T cells isolated from a healthy human donor buffy coat,
ii) providing a set of samples, wherein each sample comprises said T cells and BxPC-3, PANC-1, Ca Ski or SiHa tumor cells, and wherein the ratio of T cells to tumor cells in said sample is 4:1,
iii) adding the antibody to the set of samples at a concentration ranging from 0.0128ng/mL to 5000ng/mL (e.g., a 5-fold dilution), and incubating the samples at 37 ℃ for 72 hours,
iv) collecting 110 μ L of T cell containing supernatant from each sample and staining the T cells with fluorescently labeled antibodies to T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and with antibodies to T cell markers, such as 69-APC, CD25-PE-Cy7 and CD279/PD1-BV604 antibodies, by incubation with the antibodies at 4 ℃ for 30 minutes; and is
v) analyzing the sample by flow cytometry.
The ability to induce cytotoxicity of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells can be determined by a procedure comprising the following steps:
i) providing T cells isolated from a healthy human donor buffy coat,
ii) providing a set of test and control samples, wherein each sample comprises BxPC-3, PANC-1, Ca Ski or SiHa tumor cells and the T cells that have been allowed to adhere to the bottom of a 96-well tissue culture plate, and wherein the ratio of T cells to tumor cells in the sample is 4:1,
iii) adding the antibody to the test sample set at a concentration ranging from 0.0128ng/mL to 5000ng/mL (e.g., a 5-fold dilution) while the control sample remains untreated or is incubated with 5. mu.M staurosporine, then incubating all samples at 37 ℃ for 72 hours,
iv) incubation of adherent cells in 10% (w/w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (Resazurin) in RPMI-1640 medium supplemented with 10% (w/w) donor bovine serum containing iron and penicillin/streptomycin at 37 ℃ for 4 hours,
v) measuring the absorbance of the cells; the absorbance of cells incubated with staurosporine was set to 0% viability and the absorbance of untreated cells was set to 100% viability and the percentage of viable cells was calculated as follows:
Figure BDA0002720035660000501
the antibody of the invention may in particular be an antibody, wherein the antigen binding region capable of binding CD3 comprises:
a) heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR1-T31P + wild type VH CDR2, 3] having the sequences shown in SEQ ID NO:61, 55 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
b) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR1-T31M + wild type VH CDR2, 3] having the sequences shown in SEQ ID NO:63, 55 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
c) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR-N57E + wild type VH CDR1, 3] having the sequences shown in SEQ ID NO:54, 65 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
d) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown in SEQ ID NO:54, 55 and 67, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
e) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101N ] having the sequences shown in SEQ ID NO:54, 55 and 69, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
f) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-G105P ] having the sequences shown in SEQ ID NO:54, 55 and 71, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
g) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-S110A ] having the sequences shown in SEQ ID NO:54, 55 and 73, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
h) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-S110G ] having the sequences shown in SEQ ID NO:54, 55 and 75, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
i) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114V ] having the sequences shown in SEQ ID NO:54, 55 and 77, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
j) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114M ] having the sequences shown in SEQ ID NO:54, 55 and 79, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
k) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114R ] having the sequences shown in SEQ ID NO:54, 55 and 81 respectively and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequence shown in SEQ ID NO:58, sequence GTN and sequence shown in SEQ ID NO:59 respectively.
In certain embodiments, the antigen binding region capable of binding CD3 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild-type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild-type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
Furthermore, the present invention provides an antibody as defined above, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 6, 7 and 8 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 10, AAS and SEQ ID NO 11 respectively [059],
and
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
The invention also provides an antibody as defined above, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 41, 42 and 43 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, DAS and SEQ ID NO:46 respectively [207],
And
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
Furthermore, the present invention provides an antibody as defined above, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 48, 49 and 50, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 52, DAS and SEQ ID NO 53, respectively [226],
and
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
In the antibody of the present invention, the antigen-binding region capable of binding to human CD3 may comprise a VH sequence and a VL sequence selected from the group consisting of:
a) the VH sequence [ VH T31P ] shown by SEQ ID NO:62 and the VL sequence shown by SEQ ID NO:60,
b) a VH sequence [ VH T31M ] shown by SEQ ID NO:64 and a VL sequence shown by SEQ ID NO:60,
c) VH sequence [ VH N57E ] shown by SEQ ID NO:66 and VL sequence shown by SEQ ID NO:60,
d) VH sequence [ VH H101G ] shown by SEQ ID NO:68 and VL sequence shown by SEQ ID NO:60,
e) a VH sequence [ VH H101N ] shown by SEQ ID NO:70 and a VL sequence shown by SEQ ID NO:60,
f) VH sequence [ VH G105P ] shown by SEQ ID NO:72 and VL sequence shown by SEQ ID NO:60,
g) VH sequence [ VH S110A ] shown by SEQ ID NO:74 and VL sequence shown by SEQ ID NO:60,
h) VH sequence [ VH S110G ] shown as SEQ ID NO:76 and VL sequence shown as SEQ ID NO:60,
i) VH sequence [ VH Y114V ] shown by SEQ ID NO:78 and VL sequence shown by SEQ ID NO:60,
j) a VH sequence shown as SEQ ID NO:80 [ VH Y114M ] and a VL sequence shown as SEQ ID NO: 60; and
k) VH sequence [ VH Y114R ] shown as SEQ ID NO:82 and VL sequence shown as SEQ ID NO: 60.
Specifically, the antibody according to the present invention may be an antibody in which the antigen-binding region capable of binding to human CD3 comprises a VH sequence shown by SEQ ID NO:68 [ VH H101G ] and a VL sequence shown by SEQ ID NO: 60.
In some embodiments, the antibody according to the invention is an antibody wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:5 [059 ];
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G ] and a VL sequence shown as SEQ ID NO: 60.
In other embodiments, the antibody according to the invention is an antibody wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:40 [207 ];
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G ] and a VL sequence shown as SEQ ID NO: 60.
In other embodiments, the antibody according to the invention is an antibody wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 [226 ];
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G ] and a VL sequence shown as SEQ ID NO: 60.
As is well known to the skilled person, each antigen binding region of an antibody typically comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein each variable region comprises three CDR sequences, CDR1, CDR2 and CDR3 respectively, and four framework sequences, FR1, FR2, FR3 and FR4 respectively. This structure can also be found in antibodies according to the invention. Furthermore, an antibody according to the invention may comprise two heavy chain constant regions (CH) and two light chain constant regions (CL).
In a particular embodiment, the antibody according to the invention comprises a first and a second heavy chain, such as a first and a second heavy chain, each of which comprises at least a hinge region, a CH2 and a CH3 region. Stable heterodimeric antibodies can be obtained in high yield based on two homodimeric starting proteins containing only a few asymmetric mutations in the CH3 region, e.g. by so-called Fab arm swapping as provided in WO 2008/119353 and WO 2011/131746. Thus, in some embodiments of the invention, an antibody, a first heavy chain, wherein at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the heavy chain of human IgG1 has been substituted, and a second heavy chain, wherein at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the heavy chain of human IgG1 has been substituted, wherein said substitutions of said first and said second heavy chains are not in the same position, and wherein said amino acid positions are numbered according to EU numbering.
In particular embodiments, the invention provides antibodies wherein the amino acid in the position corresponding to K409 in the heavy chain of human IgG1 is R in the first heavy chain and the amino acid in the position corresponding to F405 in the heavy chain of human IgG1 is L in the second heavy chain, or vice versa.
In some embodiments, the antibody according to the invention comprises in addition to the antigen binding region an Fc region consisting of the Fc sequences of the two heavy chains. The first and second Fc sequences may each be of any isotype, including any human isotype, for example IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM or IgA isotype, or mixed isotypes. Preferably, the Fc region is human IgG1, IgG2, IgG3, IgG4 isotype, or a mixed isotype, e.g., human IgG1 isotype.
Antibodies according to the invention may comprise modifications in the Fc region to render the antibody inert or non-activating. Thus, in the antibodies disclosed herein, one or both heavy chains may be modified such that the antibody induces Fc-mediated effector function to a lesser extent relative to the same antibody except comprising unmodified first and second heavy chains. Fc-mediated effector function can be measured by measuring Fc-mediated CD69 expression on T cells (i.e., CD69 expression due to CD3 antibody-mediated, Fc γ receptor-dependent CD3 cross-linking), by binding to Fc γ receptors, by binding to C1q, or by inducing Fc-mediated Fc γ R cross-linking. In particular, the heavy chain constant sequence may be modified such that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type (unmodified) antibody, wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay as described in example 3 of WO 2015001085. Modification of the heavy and light chain constant sequences may also result in reduced binding of C1q to the antibody. The reduction may be at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100% compared to the unmodified antibody, and C1q binding may be determined by ELISA. Furthermore, the Fc region may be modified such that the antibody mediates a reduction in Fc-mediated T cell proliferation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% as compared to the unmodified antibody, wherein the T cell proliferation is measured in a PBMC-based functional assay.
Examples of amino acid positions that may be modified (e.g., in an IgG1 isotype antibody) include positions L234 and L235. Thus, an antibody according to the invention may comprise first and second heavy chains, and wherein in both said first and second heavy chains the amino acid residues at positions corresponding to positions L234 and L235 in the heavy chain of human IgG1 according to EU numbering are F and E, respectively.
In addition, the D265A amino acid substitution may reduce binding to all Fc γ receptors and prevent ADCC (Shields et al, 2001, J.biol.chem. (276): 6591-604). Thus, an antibody according to the invention may comprise a first and a second heavy chain, wherein in both the first and the second heavy chain the amino acid residue at a position corresponding to position D265 in the heavy chain of human IgG1 according to EU numbering is a. A further embodiment of the invention provides an antibody wherein in at least one of the first and second heavy chains, for example in both, the amino acids in the positions corresponding to positions L234, L235 and D265 in the heavy chain of human IgG1 are F, E and a, respectively. In the present application, antibodies having a combination of three amino acid substitutions L234F, L235E, and D265A and additionally the K409R or F405L mutations disclosed above are referred to with the suffix "FEAR" or "FEAL", respectively.
The amino acid sequence of the heavy chain constant region of wild-type IgG1 is identified herein as SEQ ID NO. 89. Consistent with the embodiments disclosed above, the antibodies of the present invention may comprise an IgG1 heavy chain constant region carrying the F405L substitution and having the amino acid sequence shown in SEQ ID No. 90 and/or an IgG1 heavy chain constant region carrying the K409R substitution and having the amino acid sequence shown in SEQ ID No. 94.
The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E, and D265A substitutions is identified herein as SEQ ID NO 91. The amino acid sequence of the IgG1 heavy chain constant region carrying the substitutions L234F, L235E, D265A and F405L is identified herein as SEQ ID NO 92. The amino acid sequence of the IgG1 heavy chain constant region carrying the L234F, L235E, D265A and K409R substitutions is identified herein as SEQ ID NO: 93.
The invention further provides antibodies, wherein
a) The antigen binding region capable of binding 5T4 is humanized, and/or
b) If present, the antigen binding region capable of binding to CD3 is humanized.
In addition, the invention provides antibodies, wherein
a) The antigen binding region capable of binding 5T4 is human, and/or
b) If present, the antigen binding region capable of binding CD3 is human.
In addition, the invention provides antibodies, wherein
a) The antigen binding region capable of binding 5T4 is chimeric, and/or
b) If present, the antigen binding region capable of binding CD3 is chimeric.
In some embodiments of the invention, the antibody comprises a kappa (kappa) light chain. In a particular embodiment of the invention with respect to the sequence of the bispecific antibody, the kappa light chain comprises the CDR1, -2 and-3 sequences of the 5T4 antibody light chain as disclosed above.
In a further embodiment of the invention, the antibody according to any one of the preceding claims, wherein said antibody comprises a lambda (λ) light chain. In a particular embodiment of the invention relating to bispecific antibodies, the lambda light chain comprises the CDR1, -2 and-3 sequences of the CD3 antibody light chain as disclosed above, in particular the CDR1, -2 and-3 sequences of the CD3 antibody with reduced affinity for CD3 as disclosed above. The amino acid sequence of the kappa light chain constant region is included herein as SEQ ID NO 95 and the lambda light chain constant region is included herein as SEQ ID NO 96.
In particular embodiments, the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain; for example, an antibody having a heavy chain comprising a binding region capable of binding CD3 and a lambda light chain; and a heavy chain and a kappa light chain comprising a binding region capable of binding 5T 4.
Immunoconjugates
In another aspect, the invention provides an immunoconjugate or antibody-drug conjugate (ADC) comprising an antibody as defined above, and a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic drug, a cytokine, an immunosuppressive agent, an antibiotic or a radioisotope. In general, the skilled artisan will treat a number of cytotoxic agents, chemotherapeutic drugs, cytokines, immunosuppressive agents, antibiotics, and radioisotopes, the best choice of therapeutic moiety, depending on the desired application of the immunoconjugate. For some applications, a preferred cytotoxic agent may be a microtubule disrupting agent, such as a Duostatin, such as Duostatin-3.
Nucleic acid constructs
Another aspect of the invention provides a nucleic acid construct comprising
a) A nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen binding region capable of binding 5T4 as defined hereinbefore, and/or
b) A nucleic acid sequence encoding an antibody light chain sequence comprising an antigen binding region capable of binding 5T4 as defined hereinbefore.
The nucleic acid construct may further comprise
a) A nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen binding region capable of binding CD3 as defined hereinbefore, and/or
b) A nucleic acid sequence encoding an antibody light chain sequence comprising an antigen binding region capable of binding CD3 as defined herein before.
Expression vector
Another aspect of the invention provides an expression vector comprising a nucleic acid sequence encoding a heavy and/or light chain sequence of an antibody according to the invention. In particular, the expression vector may comprise:
a) a nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen binding region capable of binding 5T4 as defined hereinbefore, and/or
b) A nucleic acid sequence encoding an antibody light chain sequence comprising an antigen binding region capable of binding 5T4 as defined hereinbefore.
The expression vector may further comprise:
a) a nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen binding region capable of binding CD3 as defined hereinbefore, and/or
b) A nucleic acid sequence encoding an antibody light chain sequence comprising an antigen binding region capable of binding CD3 as defined herein before.
In another embodiment, the expression vector further comprises a nucleic acid sequence encoding a constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody, such as human IgG1, a kappa monoclonal antibody.
In the context of the present invention, an expression vector may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-5T 4 antibody is contained in a naked DNA or RNA vector comprising, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17355-59(1997)), compacted nucleic acid vectors (e.g.as described in US 6,077,835 and/or WO 00/70087), plasmid vectors, e.g.pBR 322, pUC 19/18 or pUC 118/119, "mosquito" minimal size nucleic acid vectors (e.g.as described in Schakowski et al, Mol Ther3793-800(2001)), or as a precipitated nucleic acid vector construct, such as the precipitated construct of Cap04Constructs (as described, for example, in WO 00/46147, Benveninsty and Reshef, PNAS USA83,9551-55(1986),Wigler et al.,Cell 14725(1978) and Coraro and Pearson, solar Cell Genetics7,603(1981)). Such nucleic acid vectors and their use are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).
In one embodiment, the vector is suitable for expressing an anti-5T 4 antibody in a bacterial cell. Examples of such vectors include expression vectors, such as BlueScript (Stratagene), pIN vector Van Heeke & Schuster, J Biol Chem 264, 55035509 (1989), pET vector (Novagen, Madison Wis.), etc.
Additionally or alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters, such as alpha factor, alcohol oxidase and PGH (reviewed in F. Ausubel et al, ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience New York (1987) and Grant et al, Methods in Enzymol 153, 516544 (1987)).
The nucleic acid construct and/or vector may also comprise a nucleic acid sequence encoding a secretion/localization sequence that can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretion leaders or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., termination transit sequences, GPI anchor sequences), and the like.
In the expression vectors of the invention, the nucleic acid encoding the anti-5T 4 antibody may comprise or be associated with any suitable promoter, enhancer, and other expression promoting elements. Examples of such elements include strong expression promoters (e.g., the human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), efficient poly (a) termination sequences, origins of replication of plasmid products in e.coli, antibiotic resistance genes as selection markers, and/or convenient cloning sites (e.g., polylinkers). In contrast to constitutive promoters such as CMV IE, nucleic acids may also comprise inducible promoters (the skilled person will recognise that these terms are actually descriptors of the extent of gene expression under certain conditions).
In one embodiment, the expression vector encoding the anti-5T 4 antibody can be located in and/or delivered to a host cell or host animal by a viral vector.
Cells and host cells
In a further aspect, the invention provides a cell comprising a nucleic acid construct as defined above or an expression vector as defined above. It will be appreciated that the cell may be obtained by transfecting a host cell, such as a recombinant host cell, with the nucleic acid construct or expression vector.
The host cell may be of human origin, for example a Human Embryonic Kidney (HEK) cell, such as a HEK/Expi cell. Alternatively, it may be of rodent origin, e.g., Chinese hamster ovary cells, such as CHO/N50 cells. In addition, the host cell may be of bacterial origin.
The cell may comprise a nucleic acid sequence encoding an antibody of the invention or a portion thereof stably integrated into the genome of the cell. Alternatively, the cell may comprise a non-integrating nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence encoding for expression of an anti-5T 4 antibody or portion thereof of the invention. In particular, the host cell may comprise a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence encoding for the expression of an anti-5T 4 antibody or a portion thereof.
Composition comprising a metal oxide and a metal oxide
A further aspect of the invention provides a composition comprising an antibody; for example a bispecific antibody or immunoconjugate as defined above. The composition may be a pharmaceutical composition comprising an antibody, bispecific antibody or immunoconjugate and a pharmaceutically acceptable carrier.
The pharmaceutical compositions may be formulated according to conventional techniques, e.g., as disclosed in Remington, The Science and Practice of Pharmacy, 19 th edition, Gennaro, ed., Mack Publishing co., Easton, PA,1995, together with carriers, excipients and/or diluents and any other components suitable for pharmaceutical compositions, including known adjuvants. Pharmaceutically acceptable carriers or diluents as well as any known adjuvants and excipients should be suitable for the antibody or antibody conjugate of the invention and the mode of administration chosen. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the lack of significant negative impact on the desired biological properties of the selected compound or pharmaceutical composition of the invention (e.g., less than a substantial impact on antigen binding [ 10% or less relative inhibition, 5% or less relative inhibition, etc.).
The pharmaceutical compositions of the present invention may include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in pharmaceutical compositions.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention or its amide employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition being employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents, and the like, physiologically compatible with the compounds of the present invention.
Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils (e.g., olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil), carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organic esters such as ethyl oleate and/or various buffering agents. Other carriers are well known in the pharmaceutical art.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is contemplated.
The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, α -tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid.
The pharmaceutical compositions of the present invention may also comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the composition.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffering agents, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention can be prepared with carriers that protect the compounds from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers such as ethylene vinyl acetate alone or with waxes, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, or other materials known in the art. Methods of preparing such formulations are generally known to those skilled in the art, see, e.g., Sustained and Controlled Release Drug Delivery Systems, j.r. robinson, ed., Marcel Dekker, inc., New York, 1978.
In one embodiment, the compounds of the present invention may be formulated to ensure proper distribution in the body. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the invention is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or nonaqueous solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients as required, for example as enumerated above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients (e.g., from those enumerated above). In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any other desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any other desired ingredient from a previously sterile-filtered solution thereof.
The pharmaceutical composition of the invention may comprise an antibody, bispecific antibody or antibody-drug conjugate (ADC) of the invention, a combination of an antibody, bispecific antibody or ADC according to the invention and another therapeutic compound or a combination of a compound of the invention.
The pharmaceutical composition may be administered by any suitable route and manner. Suitable routes for administering the compounds of the invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art.
In one embodiment, the pharmaceutical composition of the invention is administered parenterally; i.e., by administration other than enteral and topical administration; typically by injection, including epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcontracting, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection, and infusion. In particular, the pharmaceutical composition of the present invention may be administered by intravenous or subcutaneous injection or infusion.
Use and therapeutic applications
The invention further provides an antibody as defined herein, e.g. a bispecific antibody, or an immunoconjugate or an antibody-drug conjugate (ADC), for use as a medicament. The anti-5T 4 antibodies or immunoconjugates of the invention can be used to treat or prevent diseases or disorders involving cells expressing 5T 4. In particular, bispecific antibodies according to the invention; that is, antibodies comprising antigen binding regions capable of binding 5T4 and CD3 may be useful in therapeutic settings where specific targeting and T cell-mediated killing of 5T 4-expressing cells is desired, and they may be more effective in certain such indications and settings than conventional anti-5T 4.
In one embodiment, disclosed herein are antibodies of the invention, e.g., bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADCs), for use in the treatment of cancer. Antibodies, such as bispecific antibodies, or immunoconjugates or antibody-drug conjugates (ADCs) are particularly useful for treating cancer, wherein the cancer is characterized by expression of 5T4 in at least some tumor cells.
The cancer may in particular be selected from kidney/kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine/endometrial/cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia. .
In addition, the invention relates to the use of an antibody according to the invention for the preparation of a medicament, e.g. a medicament for the treatment of cancer, e.g. a cancer selected from the group consisting of: kidney/kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterus/endometrium/cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia. .
In another aspect, the invention provides a method of treating a disease, the method comprising administering an antibody, immunoconjugate, composition, such as a pharmaceutical composition or antibody-drug conjugate (ADC) of the invention to a subject in need thereof.
In a particular embodiment of the invention, the method is for the treatment of cancer. The method of the invention comprises in particular the following steps:
a) selecting a subject having a cancer comprising tumor cells expressing 5T4 and/or a cancer known to express 5T 4; and
b) administering to the subject an antibody, e.g., a bispecific antibody, pharmaceutical composition or antibody-drug conjugate (ADC) of the invention.
The cancer may in particular be selected from kidney/kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterine/endometrial/cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia. .
The dosage regimen in the above-described methods and uses of treatment is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. Parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.
The effective dosage and dosage regimen of the antibody depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range of a therapeutically effective amount of a compound of the invention is about 0.001-10mg/kg, such as about 0.001-5mg/kg, for example about 0.001-2mg/kg, such as about 0.001-1mg/kg, for example about 0.001, about 0.01, about 0.1, about 1 or about 10 mg/kg. Another exemplary, non-limiting range of a therapeutically effective amount of an antibody of the invention is about 0.1-100mg/kg, such as about 0.1-50mg/kg, for example about 0.1-20mg/kg, for example about 0.1-10mg/kg, for example about 0.5, for example about 0.3, about 1, about 3, about 5, or about 8 mg/kg.
A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of antibody used in a pharmaceutical composition at a lower level than is required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, a suitable daily dose of an antibody of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Administration may be, for example, gastrointestinal External, e.g. intravenous, intramuscular or subcutaneous. In one embodiment, the composition may be prepared by mixing the components in mg/m2The calculated weekly dose was administered by infusion. Such doses may be, for example, based on the mg/kg dose provided above according to: dosage (mg/kg) x 70: 1.8. Such administration may be repeated, for example, 1 to 8 times, e.g., 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, for example 2 to 12 hours. In one embodiment, the antibody may be administered by slow continuous infusion over a long period of time (e.g., over 24 hours) to reduce toxic side effects.
In one embodiment, when given once a week, the antibody may be administered up to 8 times, e.g., 4 to 6 times, in a weekly dose calculated as a fixed dose. Such regimen may be repeated as often as desired one or more times, for example after 6 months or 12 months. Such a fixed dose may be, for example, based on the mg/kg dose provided above, with a body weight of 70kg being estimated. The dosage can be determined or adjusted by, for example, removing a biological sample and measuring the amount of the antibody of the invention in the blood after administration using an anti-idiotypic antibody targeting the 5T4 antigen-binding region of the antibody of the invention.
In one embodiment, the antibody may be administered as a maintenance therapy, such as, for example, once per week for 6 months or longer.
Antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the occurrence of events in cancer progression and/or reduce the risk of relapse upon remission of the cancer.
The antibodies of the invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition to be treated. Thus, in one embodiment, the antibody-containing medicament is for use in combination with one or more other therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent or an anti-angiogenic agent.
Antibody production
Also provided herein are methods of producing antibodies, e.g., bispecific antibodies of the invention. In particular, there is provided a method for producing an antibody of the invention comprising the steps of:
a) culturing a host cell comprising an expression vector as defined herein; and
b) and purifying the antibody from the culture medium.
In embodiments of the invention wherein the antibody comprises a binding region capable of binding 5T4 and a binding region capable of binding CD3, the antibody may be produced using a method comprising the steps of:
a) providing an antibody capable of binding 5T4 by culturing a host cell comprising an expression vector as defined herein under conditions allowing expression of the antibody capable of binding 5T4 and purifying the antibody capable of binding 5T4 from the culture medium;
b) Providing an antibody capable of binding to CD3 by culturing a host cell comprising an expression vector comprising an antibody capable of binding to CD3 under conditions that allow expression of the antibody capable of binding to CD3, and purifying the antibody capable of binding to CD3 from the culture medium
I) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined herein, and
II) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined herein;
c) incubating the antibody capable of binding 5T4 with the antibody capable of binding CD3 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and
d) obtaining the antibody.
Reagent kit
The invention further provides a kit comprising an antibody as disclosed above, e.g. for use as a companion diagnosis/for identifying within a patient population those patients having a predisposition to respond to treatment with an antibody as defined herein or an immunoconjugate or antibody-drug conjugate (ADC) as defined herein, or for predicting the efficacy of said antibody or immunoconjugate or ADC in treating patients, said kit comprising an antibody as defined herein; and instructions for use of the kit.
Anti-idiotypic antibodies
In another aspect, the present invention relates to an anti-idiotype antibody that binds to an antibody comprising at least one antigen binding region capable of binding 5T4, i.e. an antibody according to the invention as described herein. In a particular embodiment, the anti-idiotype antibody binds to an antigen binding region capable of binding 5T 4.
An anti-idiotypic (Id) antibody is an antibody that recognizes a unique determinant that is normally associated with the antigen-binding site of an antibody. anti-Id antibodies can be prepared by immunizing an animal of the same species and genetic type as the source of the anti-5T 4 monoclonal antibody with the monoclonal antibody against which the anti-Id was prepared. By generating antibodies against these idiotypic determinants (anti-Id antibodies), the immunized animal can generally recognize and respond to the idiotypic determinants of the immunizing antibody. Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the invention.
The anti-Id antibody may also be used as an "immunogen" to induce an immune response in another animal, thereby generating what is known as an anti-Id antibody. The anti-Id antibody may be epitopically identical to the original monoclonal antibody that induced the anti-Id antibody. Thus, by using antibodies directed against the idiotypic determinants of a monoclonal antibody, other clones expressing antibodies of the same specificity can be identified. The anti-Id antibody can be varied (thereby creating anti-Id antibody variants) and/or derivatized by any suitable technique, such as those described elsewhere herein for the 5T 4-specific antibodies of the invention. For example, monoclonal anti-Id antibodies can be conjugated to a carrier such as Keyhole Limpet Hemocyanin (KLH) and used to immunize BALB/c mice. Sera from these mice will typically contain anti-Id antibodies that have similar (if not identical) binding properties to the original/parent-5T 4 antibody.
Sequence of
Figure BDA0002720035660000671
Figure BDA0002720035660000681
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Figure BDA0002720035660000711
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Figure BDA0002720035660000741
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The invention is further illustrated by the following examples, which should not be construed as further limiting.
Examples
Example 1 Generation of 5T4 antibody and screening Material
Expression construct of 5T4
The following codon-optimized constructs were generated for expression of various full-length 5T4 variants: human (homo sapiens) 5T4(Uniprot accession Q13641), cynomolgus monkey (bundled monkey) 5T4(Uniprot accession Q4R8Y9) and chicken (jungle) 5T4(Uniprot accession R4GM 46). In addition, the following codon optimized constructs were generated for various 5T4 extracellular domain (ECD) variants: the ECD of human 5T4 with a C-terminal His-tag (aa 1-355 from Uniprot accession Q13641) (5T4 ECDHI) (SEQ ID NO:99), and the ECD of human 5T4 (aa 1-91) (5T4ECD 91-FcCBHis) fused to a rabbit Fc domain and a C-terminal His-tag. In SEQ ID NO 99, amino acid residues 1-31 are signal peptides; thus, the mature 5T4ECDHI protein corresponds to amino acid residues 32-363 of SEQ ID NO 99. Similarly, amino acid residues 1-31 of SEQ ID NO. 100 are signal peptides, while the mature 5T4ECD 91-FcCBHis protein corresponds to amino acid residues 32-327 of SEQ ID NO. 100.
The construct contains restriction sites suitable for cloning and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999; 234(2): 187-208). The full length human 5T4 and cynomolgus monkey 5T4 codon optimized constructs were cloned into the mammalian expression vector pcdna3.3 (Invitrogen). The full length chicken 5T4 codon optimized construct was cloned into pSB (mammalian expression vector containing Sleeping Beauty inverted terminal repeat flanking the expression cassette consisting of CMV promoter and HSV-TK polyA signal).
Generation of HEK-293F cell lines transiently expressing full-length human, cynomolgus monkey or chicken 5T4
Freestyle was obtained from Invitrogen (catalog number R790-07)TM293-F (Freestyle Medium [ HEK-293F ] adapted for suspension growth and chemical composition determination]HEK-293 subclone of (c)) and transfected with the above codon optimized construct using 293fectin (Invitrogen, catalog No. 12347-.
Purification of His-tagged 5T4
As described above, 5T4ECDHI (mature protein of SEQ ID NO: 99) was expressed in HEK-293F cells. The 5T4ECD 91-FcCBHis was expressed using the Expi293F expression platform ((Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. A14527) essentially as described by the manufacturer.
The His tag enables purification by immobilized metal affinity chromatography. In thatIn this process, the chelating agent fixed to the chromatography resin is impregnated with Co2+A cation. The supernatant containing the histidine-tagged protein was incubated with the resin in a batch mode (i.e., solution). The His-tagged protein bound strongly to the resin beads, whereas other proteins present in the culture supernatant did not bind or bind weakly compared to the His-tagged protein. After incubation, the beads were recovered from the supernatant and packed into a column. The column was washed to remove weakly bound proteins. The strongly bound His-tagged protein is then eluted with an imidazole-containing buffer that competes for His with Co 2+In combination with (1). The eluate is removed by exchanging the buffer on a desalting column.
Immunization
To generate antibodies IgG1-5T4-207 and IgG1-5T4-226, HCo17-BalbC transgenic mice (Bristol-Myers Squibb, New York, NY, USA) were immunized with 20 μ g of 5T4ECDHis protein in the Sigma adjuvant system (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. S6322) Intraperitoneally (IP) and Subcutaneously (SC) alternately at 14 day intervals. A total of 8 immunizations were performed: 4 times IP and 4 times SC.
To generate antibodies IgG1-5T4-076 and IgG1-5T4-059, transgenic mice (Bristol-Myers Squibb) of HCo12-BalbC (IgG1-5T4-076) and HCo20-BalbC (IgG1-5T4-059) were immunized alternately with IP and SC at 14-day intervals with 20 μ g of 5T4ECDHI protein in the Sigma adjuvant system. A total of 8 immunizations were performed: 4 times IP and 4 times SC.
To generate antibody IgG1-5T4-085, HCo17-BalbC transgenic mice were immunized alternately with IP and SC at 14 day intervals with 20 μ g of 5T4ECDHI protein and 20 μ g of 5T4ECD 91-FcbHis mature protein in Sigma adjuvant system. A total of 8 immunizations were performed: 4 times IP and 4 times SC.
To generate antibodies IgG1-5T4-106 and IgG1-5T4-127, HCo12-BalbC (IgG1-5T4-106) and HCo17-BalbC (IgG1-5T4-127) transgenic mice were immunized alternately with IP and SC at 14-day intervals with 20 μ g of 5T4ECD 91-FcCBHis mature protein in the Sigma adjuvant system. A total of 8 immunizations were performed: 4 times IP and 4 times SC.
Mice with at least two consecutive 5T 4-specific antibody titers in the antigen-specific screening fluorometric micro-volume assay technique (FMAT) as described below were boosted with 10 μ g5T 4ECDHis or 10 μ g5T4ECD91-FcRbHis (in intravenously injected PBS), and spleen and lymph node cells of these mice were fused 3-4 days later.
Homogeneous antigen-specific screening assays
The presence of 5T4 antibody in the immunized mouse serum or HuMAb (human monoclonal antibody) hybridoma or transfectoma culture supernatants was determined by a homogeneous antigen-specific screening assay using FMAT (Applied Biosystems, Foster City, CA, USA). For this, a combination of 4 cell-based assays was used.
Serum from immunized mice or samples of hybridoma or transfectoma culture supernatants were analyzed for binding of human antibodies to HEK-293F cells transiently expressing human 5T4, HEK-293F cells transiently expressing cynomolgus monkey 5T4, streptavidin-coated polystyrene particles (0.5% w/v; 6.7 μm; Spherotech, Lake Forest, IL, USA, Cat. SVP-60-5) coated with 5T4ECD 91-FcBHis, and HEK-293 wild-type cells (negative control).
The sample was added to the cells to allow binding of 5T 4. Subsequently, a fluorescent conjugate (Affinipure goat anti-human IgG Fc) was used
Figure BDA0002720035660000791
647; jackson ImmunoResearch, catalog number 109-. IgG1-5T4-H8-F405L was used as a positive control, and Chrompure human IgG whole molecule (Jackson ImmunoResearch, catalog # 009-. The samples were scanned using ImageXpress vessels (Molecular devices, LLC, Sunnyvale, Calif., USA) and the total fluorescence was used as a reading. A sample is described as positive when the count is above 50 and the count x fluorescence is at least three times higher than the negative control.
Generation of HuMAb hybridomas
HuMAb mice with sufficient antigen-specific titer development were sacrificed (as described above) and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Spleen cells and lymph node cells were fused by electrofusion with a mouse myeloma cell line (SP2.0 cells) using the CytoPulse CEEF 50 electrofusion system (celectis, Paris, France) essentially according to the manufacturer's instructions. Next, antigen-positive primary wells were subcloned using the ClonePix system (Genetix, Hampshire, UK). To this end, specific primary well hybridomas were inoculated into a semi-solid medium made of 40% Clone Media (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA). Subclones were retested for 5T4 binding according to the antigen-specific binding assay and scanned using the IsoCyte system (Molecular Devices) as described above. IgG levels were measured using the Octet system (Fortebio, Menlo Park, USA) in order to select the best producing clone per primary well for further amplification. The resulting HuMAb hybridomas are further expanded and cultured based on standard Protocols (e.g., as described in Coligan j.e., Bierer, b.e., Margulies, d.h., Shevach, e.m.and Strober, w. compiled Current Protocols in Immunology, John Wiley & Sons, inc., 2006).
Sequence analysis of 5T4 antibody variable domains and cloning in expression vectors
From 2 to 5x106Total RNA was prepared from each hybridoma cell and 5'-RACE complementary DNA (cDNA) was prepared from 100ng of total RNA using the SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. The VH and VL coding regions were amplified by PCR and cloned in-frame directly into the p33G1f and p33Kappa expression vectors (pcDNA3.3-based vectors with codon optimized human IgG1m (f) and Kappa constant domains, respectively) by ligation independent cloning (Aslantis, C.and P.J.de Jong, Nucleic Acids Res 1990; 18(20): 6069-74). The variable domains from these expression vectors were sequenced and the CDRs annotated according to the IMGT definition (Lefranc MP. et al, Nucleic Acids Research,27,209-212,1999and Brochet X. Nucleic Acids Res.36, W503-508 (2008)). Clones with the correct Open Reading Frame (ORF) were expressed and tested for binding to antigen. The leader group was ordered with codon-optimized sequences (GeneArt, Thermo Fisher Scientific) and generated using the Expi293 expression system according to the manufacturer's instructions (Thermo Fisher Scientific). The antibodies in these supernatants were purified and used for functional characterization. The sequences of the resulting leader clones are shown in the table above.
5T4 control antibody
In some embodiments, a comparison antibody against 5T4 (IgG1-5T4-H8, IgG1-5T4-A3, and IgG1-5T4-a1) previously described in WO2007/106744 is used. Codon-optimized antibody coding sequences were synthesized and cloned into pcdna3.3 expression vector (Thermo Fisher Scientific).
IgG1-b12 antibodies
In some embodiments, antibody b12, an HIV-1gp 120-specific antibody (Barbas, CF. J Mol biol.1993Apr 5; 230(3):812-23), was used as a negative control. The codon optimized antibody coding sequence for this control antibody was synthesized and cloned into the pcdna3.3 expression vector (Thermo Fisher Scientific). Sequences of the heavy chain Variable (VH) region and the light chain Variable (VL) region are included herein as SEQ ID NOS: 97 and 98, respectively.
Example 2 measurement of binding affinity of 5T 4-specific antibodies Using biolayer interferometry
The affinity of the 5T4 antibody for recombinant 5T4 protein was determined on an Octet HTX instrument (ForteBio, Portsmouth, UK) using label-free biolayer interferometry. The 5T4 antibody (1. mu.g/mL) was immobilized on an anti-human IgG Fc capture biosensor (ForteBio) for 600 seconds. After baseline measurements (100s), association (200s) and dissociation (1000s) of human 5T4ECDHI (mature protein of SEQ ID NO: 99) or recombinant cynomolgus 5T4 protein (Cusabio; catalog number CSB-MP024093MOV) in sample dilutions (ForteBio) were determined using a 2-fold dilution series (ranging from 100nM to 1.56nM) starting at 3.58. mu.g/mL (100nM) of human 5T4ECDHI or 3.99. mu.g/mL (100nM) of cynomolgus 5T4 with shaking at 1000rpm at 30 ℃. Data were analyzed using data analysis software v9.0.0.12 (ForteBio). Separately for each antibody, the value of the reference well containing only the sample diluent during the association and dissociation steps was subtracted from the value of the well containing the antigen. The Y-axis was aligned to the last 10s of the baseline and the inter-step corrected alignment to dissociation and Savitzky-Golay filtering was applied. <The 0.05nm response was excluded from the analysis. The data were fitted using a 1:1 model and a global full fit (global full fit) with 200s association time and 1000s or 50s dissociation time as windows of interest. Default use with complete dissociationTime (1000s) was used as a fit to the window of interest. Based on R2Visual inspection of values and fit, dissociation time of 50s was used as a window of interest for IgG1-5T 4-127-FEAR.
Table 1 shows the association rate constant k of the 5T4 antibody for human 5T4ECDH as determined by biolayer interferometrya(1/Ms), dissociation rate constant kd(1/s) and equilibrium dissociation constant KD(M). The range of affinity of the antibody for human 5T4 was measured and ranged from 1.3X10-9–2.7x10-8And M. The response of IgG1-5T4-085-FEAR was below 0.05nm, which prevented correct fitting of the data (lower R for these fits)2Value). Furthermore, the response of IgG1-5T4-076-FEAR could not be correctly fitted. These data are shown in italics.
Table 2 shows the association rate constant k of cynomolgus monkey 5T4 as determined by biolayer interferometrya(1/Ms), dissociation rate constant kd(1/s) and equilibrium dissociation constant KD(M). Measuring the affinity range of the antibody to cynomolgus monkey 5T4, the range is 1.1x10-9–4.1x10-8And M. The responses of IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR were below 0.05nm, which prevented correct fitting of the data (lower R for these fits) 2Value). Furthermore, the response of IgG1-5T4-076-FEAR could not be correctly fitted. These data are shown in italics.
Table 1: binding affinity of monospecific bivalent 5T4 antibody to human 5T4 extracellular domain as determined by label-free biolayer interferometry.
Figure BDA0002720035660000811
Figure BDA0002720035660000821
Table 2: binding affinity of the monospecific bivalent 5T4 antibody to the extracellular domain of cynomolgus monkey 5T4 as determined by label-free biolayer interferometry.
Figure BDA0002720035660000822
Example 3 Cross-blocking of 5T4 antibody by biolayer interferometry
Antibody cross-blocking analysis (epitope binding) was performed on an Octet HTX instrument (ForteBio) using biolayer interferometry. The 5T4 antibody (20 μ g/mL in 10mM sodium acetate buffer, pH 6.0, ForteBio) was immobilized on an amine reactive second generation (AR2G) biosensor (ForteBio) according to the manufacturer's instructions. After baseline measurements (100s) in sample dilutions (ForteBio), the immobilized antibody-containing biosensor was loaded with 100nM (3.6. mu.g/mL) of human 5T4ECDHis (mature protein of SEQ ID NO: 99) for 500 s. Next, the association response of a second 5T4 antibody (10. mu.g/mL) was determined for 500 s. The biosensor was regenerated by 3 exposures of 5s to 10mM glycine pH 2.5 followed by sample dilutions and measurements repeated starting from the baseline step with a new set of second 5T4 antibodies. Each biosensor was used up to four times. Measurements were made at 30 ℃ using a shaker speed of 1000 rpm. Data were analyzed using data analysis software v9.0.0.12 (ForteBio). The Y-axis was aligned to the association step and filtered using Savitzky-Golay. The response of the sample diluent in the association step was subtracted from the association response of the second antibody to correct for dissociation of the 5T4ECDHis from the immobilized antibody. The corrected association response is plotted in matrix form. Typically, responses >0.1nm are considered non-blocking antibody pairs (white), while responses between-0.1 and 0.1nm are considered blocking antibody pairs (dark grey). For some antibody pairs, the secondary antibody showed an initial positive response followed by a decrease in signal. This is considered to be an antibody displacement (light grey), i.e. a second antibody displaces the interaction between the primary antibody and the antigen (Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morisige W, et al (2017) Antibodies Targeting Closel Adjacent or Minimally Overlapping Epitopes Can One animal plos ONE 12(1): e0169535.doi: 10.1371/joural. point. p. 0169535). In some cases, the data curves require visual inspection by an expert to assign blocking, non-blocking or displacement properties to antibody pairs.
Cross-blocking experiments were performed on antibodies IgG1-5T4-059-FEAR, IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-127-FEAR, IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, and prior art antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L and IgG1-5T 4-A3-F405L. The results are summarized in table 3.
None of the antibodies (except IgG1-5T4-A1-F405L itself) blocked the binding of IgG1-5T4-A1-F405L to 5T4 ECDHI. Antibodies IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, IgG1-5T4-127-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR (as well as IgG1-5T4-H8-FEAR itself) block the binding of IgG1-5T4-H8-FEAR to 5T4 ECDHI. Antibodies IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR and IgG1-5T4-127-FEAR (as well as IgG1-5T4-A3-F405L per se) also blocked the binding of IgG1-5T4-A3-F405L to 5T4ECDHI, while antibodies IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR did not block the binding of IgG1-5T4-A3-F405L to 5T4 ECDHI. Antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR in combination with IgG1-5T4-A3-F405L (which is described in more detail in example 4) show antibody substitutions.
Table 3: antibody cross-blocking as determined by biolayer interferometry.
The first column shows the immobilized antibody and the first row shows the antibody in solution. The corrected association response of the antibody in solution is shown. Cross-blocking of antibodies is indicated by dark grey, and replacement antibody combinations by light grey and asterisks. The non-blocking antibody combination was unlabeled (transparent background).
Figure BDA0002720035660000841
Example 4 combination of IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR with IgG1-5T4-A3-F405L Antibody replacement with IgG1-5T4-226-FEAR
Displacement of the antibody was demonstrated using a biolayer interferometer on an Octet HTX instrument (ForteBio). IgG1-5T4-A3-F405L (20. mu.g/mL in 10mM sodium acetate buffer pH 6.0, ForteBio) was immobilized on an amine reactive second generation (AR2G) biosensor (ForteBio) according to the manufacturer's instructions. After baseline measurements (100s) in sample dilutions (ForteBio), biosensors containing immobilized IgG1-5T4-A3-F405L antibody were loaded with human 5T4ECDHis (mature protein of SEQ ID NO: 99) 100nM (3.6. mu.g/mL) for 500 s. Next, the association response of a second 5T4 antibody (IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T 4-226-FEAR; 10. mu.g/mL) or sample dilution (buffer control) was determined for 500 s. The experiment was carried out at 30 ℃ using an oscillator speed of 1000 rpm. Data were analyzed using data analysis software v9.0.0.12 (ForteBio). The buffer control response was subtracted from the response of the secondary antibody to correct for dissociation of human 5T4ECDHis from immobilized IgG1-5T4-A3-F405L, align the Y-axis with the association step, and filter using Savitzky-Golay.
As shown in FIG. 1, IgG1-5T4-A3-F405L showed no binding, indicating cross-blocking (self-blocking) with IgG1-5T 4-A3-F405L. IgG1-5T4-H8-FEAR showed binding to 5T4ECDHI and thus no cross-blocking with IgG1-5T 4-A3-F405L. IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR initially showed positive responses (indicating cross-blocking with the IgG1-5T4-A3-F405L-5T4ECDHis complex rather than with IgG1-5T 4-A3-F405L), and then the response decreased, which decreased to the self-blocking response of IgG1-5T 4-A3-F405L. This demonstrates the mass loss from the IgG1-5T4-A3-F405L-5T4ECDHI complex, indicating dissociation of human 5T4ECDHI from IgG1-5T4-A3-F405L following induction of the binding complex by IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR, and IgG1-5T 4-226-FEAR. This phenomenon is described as antibody displacement and indicates that Epitopes are Closely Adjacent or Minimally Overlapping (Abdiche YN, Yeung AY, Ni I, Stone D, Miles A, Morisige W, et al (2017) Antibodies Targeting CloselJacment or Minimally Overlapping Epitopes Can One antenna One 12(1): e6950135. doi: 10.1371/joural. po. 0169535)). This indicates that antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind different epitopes on 5T4 compared to IgG1-5T 4-A3-F405L.
Example 5-Simultaneous binding of 5T4 antibody to Membrane-bound 5T4 measured by flow cytometry
The binding of IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR antibodies to membrane-bound 5T4 in the presence of IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L was assessed by flow cytometry. IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR were conjugated to fluorescein isothiocyanate (FITC, Thermo Fisher Scientific) according to the manufacturer's instructions. SK-OV-3 cells (50,000 cells per condition) expressing approximately 20,000 molecules/cell of 5T4 were incubated with a mixture of 10. mu.g/mL unconjugated 5T4 antibody (IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12, IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR) and 2. mu.g/mL FITC conjugated 5T4 antibody (IgG1-5T4-H8-FEAR-FITC, IgG1-5T 4-207-AR-FITC and IgG1-5T 4-226-FEAR-FITC). Table 4 shows a summary of the combinations tested. After incubation at 4 ℃ for 30 minutes, the cells were centrifuged at 1200RPM for 5 minutes and the supernatant was discarded. Cells were resuspended in 100. mu.L FACS buffer supplemented with 1:4000 Topro-3-iododine (molecular probes). The Mean Fluorescence Intensity (MFI) of the FITC signal was measured using a flow cytometer (FACS Fortessa, BD Biosciences). Percent binding was calculated using the following formula:
([ MFI of cells with Ab-FITC and unconjugated antibody-without Ab-FITC or unconjugated antibody MFI of cells]*100)(MFI of cells with Ab-FITC and isotype control-MFI of cells without Ab-FITC or unconjugated antibody)
FIG. 2 shows that the binding of IgG1-5T4-H8-FEAR-FITC, IgG1-5T4-207-FEAR-FITC, and IgG1-5T4-226-FEAR-FITC is blocked in the presence of their unconjugated counterparts. However, in the presence of unconjugated IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L or IgG1-b12, binding of IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC to membrane bound 5T4 was still observed and was comparable to binding of IgG1-5T4-H8-FEAR-FITC to membrane bound 5T4 in the presence of unconjugated IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L or IgG1-b 12. Antibodies IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind to different epitopes on 5T4 compared to antibodies IgG1-5T4-A1-F405L and IgG1-5T 4-A3-F405L.
Table 4: summary of antibody combinations used in flow cytometry experiments.
FITC-labeled antibody (2. mu.g/mL) Unconjugated antibody (10. mu.g/mL)
1 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-H8-FEAR
2 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A3-F405L
3 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-207-FEAR
4 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-226-FEAR
5 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A1-F405L
6 IgG1-5T4-H8-FEAR-FITC IgG1-b12
7 IgG1-5T4-207-FEAR-FITC IgG1-5T4-H8-FEAR
8 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A3-F405L
9 IgG1-5T4-207-FEAR-FITC IgG1-5T4-207-FEAR
10 IgG1-5T4-207-FEAR-FITC IgG1-5T4-226-FEAR
11 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A1-F405L
12 IgG1-5T4-207-FEAR-FITC IgG1-b12
13 IgG1-5T4-226-FEAR-FITC IgG1-5T4-H8-FEAR
14 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A3-F405L
15 IgG1-5T4-226-FEAR-FITC IgG1-5T4-207-FEAR
16 IgG1-5T4-226-FEAR-FITC IgG1-5T4-226-FEAR
17 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A1-F405L
18 IgG1-5T4-226-FEAR-FITC IgG1-b12
Example 6-5T4 binding of antibodies to HEK-293 cells transfected with human or Chicken 5T4
Binding of the 5T4 antibody to HEK-293 cells transiently transfected with full-length human or chicken 5T4 (produced as described in example 1) was analyzed by flow cytometry. Cells (5X 10) were incubated at 4 deg.C 4Individual cells/well) were incubated in polystyrene 96-well round bottom plates (Greiner bio-one, catalog No. 650180) for 30 minutes with serial dilutions (ranging from 0.01 to 10 μ g/mL, in 3-fold dilution steps) of 5T4 antibody in 50 μ L PBS/0.1% BSA/0.02% azide (staining buffer). After washing twice in staining buffer, cells were washed in 50. mu.L of goat anti-human IgG F conjugated with R-Phycoerythrin (PE) (ab')2(1: 500; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa., Cat. No. 109-. Cells were washed twice in staining buffer, resuspended in 20 μ L staining buffer, and analyzed on an iQue screener (Intellicy Corporation, USA). Binding curves were analyzed by non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 3A shows dose-dependent binding of IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR and IgG1-5T4-A3-F405L to HEK-293 cells transfected with full-length human 5T 4. FIG. 3B shows that while dose-dependent binding of IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR to HEK-293 cells transfected with full-length chicken 5T4 was observed, IgG1-5T4-A3-F405L showed minimal binding to HEK-293 cells transfected with full-length chicken 5T 4. The negative control antibody IgG1-b12-K409R did not show binding to HEK-293 cells transfected with full-length human or chicken 5T4 at a concentration of 10. mu.g/mL.
Example 7-5 internalizing ability of T4 antibody in tumor cells
Experiments were performed to characterize the internalizing ability of the monovalent 5T4 antibody. Intracellular payload delivery and resulting cytotoxicity was used as a readout for internalization of the 5T4 antibody upon target binding. Toxin-conjugated bispecific antibodies were generated by controlled Fab arm exchange of the unconjugated 5T4 antibody with an IgG1-b12 antibody (HIV-1gp120 specific) that has been conjugated with the microtubule disrupting agent Duostatin-3, which antibody recognizes 5T4 with one Fab arm and an unrelated antigen (HIV-1gp120, which is not expressed on tumor cells) with the second Fab arm. The resulting bispecific Duostatin-3 conjugated antibodies carried 1 toxin molecule per antibody (drug-antibody ratio 1). Serial dilutions (0.00152-10. mu.g/mL, 3-fold) of Duostatin-3 conjugated bispecific antibody that binds monovalent to 5T4 were added to MDA-MB-468 (breast cancer cell line, ATCC, HTB-132 clone) or HCC1954 (breast cancer cell line, clone CRL-2338) cells seeded in flat-bottom 96-well tissue culture plates (5,000 cells/well; Greiner-bio-one, The Netherlands, Cat. No. 655180). Cells were incubated at 37 ℃ for 5 days and then cell viability was assessed using the CellTiter-Glo luminescent cell viability assay (Promega, USA, Cat. No. G7570) according to the manufacturer's instructions. Cytotoxicity curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 4 shows the cytotoxic ability of Duostatin-3 conjugated bispecific antibodies that bind 5T4 monovalent in MDA-MB-468(A) or HCC1954 cells (B). BsIgG1-5T4-H8-FEARxb12-vcDuo3 is highly capable of inducing cytotoxicity, indicating the potent internalizing ability of the antibody. In contrast, bsIgG1-5T4-076-FEARxb12-vcDuo3, bsIgG1-5T4-085-FEARxb12-vcDuo3 and bsIgG1-5T4-127-FEARxb12-vcDuo3 did not induce any cytotoxicity; the dose response curve was similar to that of the non-binding IgG1-b12-vcDuo3 control antibody. This indicates poor internalization of those antibodies upon binding to membrane-bound 5T 4. BsIgG1-5T4-059-FEARxb12-vcDuo3, bsIgG1-5T4-106-FEARxb12-vcDuo3, bsIgG1-5T4-207-FEARxb12-vcDuo3 and bsIgG1-5T4-226-FEARxb12-vcDuo3 induced moderate cytotoxicity in both test cell lines, indicating that these monovalent 5T4 antibodies induced internalization, but to a lesser extent than bsIgG1-5T4-H8-FEARxb12-vcDuo 3.
Example 8-humanized CD3 antibody for the production of CD3x5T4 bispecific antibody
The production of the humanized antibody IgG1-huCD3-H1L1 is described in example 1 of WO 2015/001085. IgG1-huCD3-H1L1 is referred to herein as "IgG 1-huCD 3". The antibody IgG1-huCD3-H1L1-FEAL is a variant thereof which, in addition to mutations that allow the generation of bispecific antibodies by controlled Fab arm exchange: L234F, L235E, D265A and F405L have in addition to the Fc domain amino acid substitutions that prevent interaction with IgG Fc receptors (Fc gamma receptors [ Fc γ R ]) and complement, as described above. It has previously been demonstrated that these mutations have no effect on the binding of the antibodies into which they are introduced to the target (see, e.g., US 2015/0337049).
The production of the humanized antibody IgG1-huCD3-H1L1-H101G is described in example 2 of WO 2017/009442. IgG1-huCD3-H1L1-H101G is referred to as "IgG 1-huCD 3-H101G". As described above, antibody IgG1-huCD3-H101G-FEAL is a variant with amino acid substitutions L234F, L235E, D265A and F405L.
Example 9 determination of CD3 binding affinity Using biolayer interferometry
The binding affinity of selected CD3 antibodies (including IgG1-huCD3 and IgG1-huCD3-H101G) was determined as described in example 7 of WO 2017/009442.
Briefly, the binding affinity of selected CD3 antibodies in the form of IgG1-huCD3-FEAL to recombinant soluble CD3(CD3E27-GSKa) (mature protein of SEQ ID NO: 101) was determined using biolayer interferometry on a ForteBio Octet HTX (ForteBio). An anti-human Fc capture biosensor (ForteBio, Cat. No. 18-5060) was loaded with hlgG (1mg/mL) for 600 s. After baseline measurement (200s), the CD3E27-GSKa association (1000s) and dissociation (2000s) were determined using a CD3E27-GSKa concentration range (sample dilution, ForteBio, Cat. No. 18-5028) of 27.11. mu.g/mL-0.04. mu.g/mL (1000nM-1.4nM) with a three-fold dilution step. For the calculation, the theoretical molecular weight of CD3E27-GSKa based on the amino acid sequence, i.e., 27.11kDa, was used. The experiment was performed with shaking at 1000rpm at 30 ℃. Each antibody was tested in at least two independent experiments. Data were analyzed using ForteBio data analysis software v8.1, using a 1:1 model and a global full fit with an association time of 1000s and a dissociation time of 100 s. The data trace was corrected by subtracting the reference curve (antibody on biosensor, measured using only sample diluent), aligning the Y-axis to the last 10s of baseline, and applying the inter-step correction and Savitzky-Golay filtering. Data traces with response <0.05nm were excluded from the analysis.
Table 5 shows the association rate constant k of recombinant CD3 as determined by biolayer interferometrya(1/Ms), dissociation rate constant kd(1/s) and equilibrium dissociation constant KD(M). With IgG1-huCD3-H101G-FEAL (K)D638nM), IgG1-huCD3-FEAL showed relatively high binding affinity (K) for recombinant CD3D：15nM)。
Table 5: binding affinity of monospecific bivalent CD3 antibody to recombinant CD3 as determined by label-free biolayer interferometry
Figure BDA0002720035660000891
Example 10 bispecific antibody Generation by 2-MEA induced Fab arm exchange
Such as WO2011147986, WO2011131746 and WO2013060867(Genmab) and Labrijn et al (Labrijn et al, PNAS 2013,110: 5145-50; Graner et al, MAbs 2013,5:962973) said, use
Figure BDA0002720035660000892
The platform technology, 2-MEA induced Fab-arm exchange, generates bispecific antibodies in vitro. To produce bispecific antibodies by this approach, an IgG1 molecule carrying a single mutation in the CH3 domain was generated: in one parent IgG1 antibody was the F405L mutation (i.e., CD3 antibody) and in the other parent IgG1 antibody was the K409R mutation (i.e., 5T4 or control, HIV-1gp120 specific antibody). In addition to these mutations, the parent IgG1 antibody comprises substitutions in the Fc domain that result in a failure to interact with the IgG Fc receptor (Fc gamma receptor) and complement: L234F, L235E, D265A (FEA).
To generate bispecific antibodies, the two parent antibodies were added in equal amounts by mass in PBS buffer (phosphate buffered saline; 8.7mM HPO)4 2-,1.8mM H2PO4 -,163.9mM Na+,140.3mM Cl-pH 7.4). 2-mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75mM and the reaction mixture was incubated at 31 ℃ for 5 hours. According to the manufacturer's protocol, Slide-a-Lyzer sledge (carriages) with a molecular weight cut-off of 10kDa (Thermo Fisher Scientific) was used to remove 2-MEA by dialysis into PBS buffer to allow reoxidation of interchain disulfide bonds and formation of intact bispecific antibodies.
The following antibodies were used in the examples:
CD3 antibody
IgG1-huCD3-FEAL (with VH and VL sequences shown as SEQ ID NO:57 and SEQ ID NO: 60).
IgG1-huCD3-H101G-FEAL (having VH and VL sequences shown in SEQ ID NO:68 and SEQ ID NO: 60)
5T4 antibody
IgG1-5T4-207-FEAR (having VH and VL sequences shown as SEQ ID NO:40 and SEQ ID NO: 44)
IgG1-5T4-226-FEAR (having VH and VL sequences shown in SEQ ID NO:47 and SEQ ID NO: 51)
IgG1-5T4-059-FEAR (having VH and VL sequences shown in SEQ ID NO:5 and SEQ ID NO: 9)
IgG1-5T4-076-FEAR (having VH and VL sequences shown in SEQ ID NO:12 and SEQ ID NO: 16)
IgG1-5T4-085-FEAR (having VH and VL sequences shown in SEQ ID NO:19 and SEQ ID NO: 23)
IgG1-5T4-106-FEAR (having VH and VL sequences shown as SEQ ID NO:26 and SEQ ID NO: 30)
IgG1-5T4-127-FEAR (having VH and VL sequences shown in SEQ ID NO:33 and SEQ ID NO: 37)
IgG1-5T4-H8-FEAR (based on the 5T4 antibody H8 from Wyeth (WO 2007/106744 and US 2010/0173382); having VH and VL sequences shown in SEQ ID NO:87 and SEQ ID NO: 88)
IgG1-5T4-A1-F405L (based on the 5T4 antibody A1 from Wyeth (WO 2007/106744 and US 8044178); having VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO: 84)
IgG1-5T4-A1-FEAR (based on the 5T4 antibody A1 from Wyeth (WO 2007/106744 and US 8044178); having VH and VL sequences shown in SEQ ID NO:83 and SEQ ID NO: 84)
IgG1-5T4-A3-F405L (based on the 5T4 antibody A3 from Wyeth (WO 2007/106744 and US 8759495); having VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO: 86)
IgG1-5T4-A3-FEAR (based on the 5T4 antibody A3 from Wyeth (WO 2007/106744 and US 8759495); having VH and VL sequences shown in SEQ ID NO:85 and SEQ ID NO: 86)
Bispecific antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR
bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR
bsIgG1-huCD3-H101G-FEALxb12-FEAR
bsIgG1-huCD3-FEALx5T4-207-FEAR
bsIgG1-huCD3-FEALx5T4-226-FEAR
bsIgG1-huCD3-FEALx5T4-059-FEAR
bsIgG1-huCD3-FEALx5T4-106-FEAR
bsIgG1-huCD3-FEALx5T4-H8-FEAR
bsIgG1-huCD3-FEALx5T4-A1-FEAR
bsIgG1-huCD3-FEALx5T4-A3-FEAR
bsIgG1-b12-FEALx5T4-207-FEAR
Fluorescein Isothiocyanate (FITC) -labeled bispecific antibody
bsIgG1-b12-FEALx5T4-059-FEAR-FITC
bsIgG1-b12-FEALx5T4-207-FEAR-FITC
bsIgG1-b12-FEALx5T4-226-FEAR-FITC
bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC
bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC
Bispecific antibody conjugated with Duostatin-3
BsIgG1-5T4-H8-FEARxb12-vcDuo3
bsIgG1-5T4-076-FEARxb12-vcDuo3
bsIgG1-5T4-085-FEARxb12-vcDuo3
bsIgG1-5T4-127-FEARxb12-vcDuo3
BsIgG1-5T4-059-FEARxb12-vcDuo3
bsIgG1-5T4-106-FEARxb12-vcDuo3
bsIgG1-5T4-207-FEARxb12-vcDuo3
bsIgG1-5T4-226-FEARxb12-vcDuo3。
Non-binding control antibody
IgG-b12 is an HIV-1gp 120-specific antibody (Barbas, CF. J Mol biol.1993Apr 5; 230(3):812-23) that is used in some instances as the negative, non-binding, control second arm of bispecific antibodies.
IgG1-b12-F405L is a variant thereof with the substitution F405L.
IgG1-b12-FEAL is a mutation thereof in allowing the generation of bispecific antibodies by controlled Fab arm exchange: variants with substitutions in addition to L234F, L235E, D265A and F405L that result in Fc domains that are unable to interact with IgG Fc receptors (Fc gamma receptors) and complement.
IgG1-b12-K409R is a variant thereof with the substitution K409R.
IgG1-b12-FEAR is a mutation thereof that allows the generation of bispecific antibodies by controlled Fab arm exchange: variants other than L234F, L235E, D265A and K409R having substitutions that result in Fc domains that are unable to interact with IgG Fc receptors (Fc gamma receptors) and complement.
Example 11 binding of CD3x5T4 bispecific antibody to cynomolgus monkey and human 5T4 expressed in HEK-293 cells
Binding of bispecific monovalent CD3x5T4 antibody and monospecific bivalent 5T4 antibody to plasma membranes of HEK-293 cells (generated as described in example 1) transiently transfected with human 5T4 or cynomolgus monkey (cynomolgus monkey) 5T4 was analyzed by flow cytometry.
Cells (3X 10) were incubated at 4 deg.C4Individual cells/well) were incubated in a polystyrene 96-well round bottom plate (Greiner bio-one, catalog No. 650180) for 30 minutes with 100 μ L of serial dilutions (ranging from 0.0137 to 10 μ g/mL, in 3-fold dilution steps) of the antibody in PBS/0.1% BSA/0.02% azide (staining buffer). The experiments were performed in technical replicates. After washing twice in staining buffer, cells were incubated in 50 μ L of secondary antibody for 30 min at 4 ℃. As secondary antibody, FITC-conjugated goat anti-human IgG F (ab') diluted 1:200 in staining buffer was used in all experiments 2(Southern Biotech, USA, Cat. No. 2043-02). Cells were washed twice in staining buffer, resuspended in 30 μ L staining buffer, and analyzed on an iQue Screen (Intellicy Corporation, USA). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 5(I) (left panel) shows bispecific antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR(A),
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR(B),
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (C) and
bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR (D) (monovalent binding to 5T4)
Shows dose-dependent binding to HEK-293 cells transfected with human 5T4, comparable to the binding of the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR and IgG1-5T4-H8-FEAR, respectively.
FIG. 5(I) (right panel) shows bispecific antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR(A),
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (B) and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (C) (which bind monovalent to 5T4)
Show dose-dependent binding to HEK-293 cells transfected with cynomolgus monkey 5T4, comparable to the binding of the monospecific bivalent 5T4 antibody IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR, respectively.
BsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T4-H8-FEAR showed poor binding to cynomolgus monkey 5T4, consistent with the experiments described in example 2 and WO 2007/106744. As a negative control, IgG1-b12-K409R (3. mu.g/mL) was included in these experiments and the antibody showed no binding to HEK-293 cells transfected with human or cynomolgus monkey 5T 4.
In a second experiment, staining was performed with minor adjustments as described above. Cells were incubated with serial dilutions of antibody ranging from 0.000128 to 10 μ g/mL in 5-fold dilution steps. As secondary antibody, Phycoerythrin (PE) -conjugated goat anti-human IgG F (ab') 2(Jackson Immunoresearch, UK, Cat. No. 109-116-098) was used at a 1:200 dilution in staining buffer.
FIG. 5(II) shows that antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR (A), bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR (B), bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG 4-5T 4-059-FEAR (C), bsIgG 4-huCD 4-H101 4-ALFEx 5T4-106-FEAR IgG and IgG 36127-5T 4-106-FEAR, (bsIgG FEAR 4-FEAR 4-FEX 5T 4-FEAR 360772-FEAR) and IgG 365-36085-4-FEASH-FEAR and IgG-36085-4-FEX 4-FEAR (C), (bsIgG 4-FEH-4-FEH-36085-4-FEH-4-FEH-36, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR (H), bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR (I) showed dose-dependent binding to HEK-293 cells transfected with human 5T4 (left panel) and HEK-293 cells transfected with cynomolgus monkey 5T4 (right panel). Likewise, the binding curves for bivalent monospecific and bispecific monovalent antibodies show a similar trend between human and cynomolgus 5T 4.
Example 12 binding of CD3x5T4 bispecific antibody to 5T4 Positive human tumor cells
The binding of the CD3x5T4 bispecific antibody to the human tumor cell line HeLa (cervical adenocarcinoma; ATCC, cat # CCL-2) and MDA-MB-231 (breast carcinoma; ATCC, cat # HTB-26) expressing 5T4 was analyzed by flow cytometry. Neither HeLa nor MDA-MB-231 cells expressed CD 3.
Cells (3X 10) were incubated at 4 deg.C4Individual cells/well) were incubated in a polystyrene 96-well round bottom plate (Greiner bio-one, catalog No. 650180) for 30 minutes with 100 μ L of a serial dilution of the antibody in PBS/0.1% BSA/0.02% azide (staining buffer), ranging from 0.000152 to 3 μ g/mL, at a 3-fold dilution step. After washing twice in staining buffer, cells were incubated in 50 μ L of secondary antibody for 30 min at 4 ℃. As the secondary antibody, Fluorescein Isothiocyanate (FITC) -conjugated goat anti-human IgG F (ab') diluted at 1:400 in staining buffer was used2(Southern Biotech, USA, Cat. No. 2043-02) A first experiment was performed. Next, the cells were washed twice in staining buffer, resuspended in 120 μ L staining buffer, and analyzed on BD LSRFortessa FACS (BD Biosciences, USA). Binding curves were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 6(I) (left panel) shows that the CD3x5T4 bispecific antibody bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (A) and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (B) show higher dose dependent binding to HeLa cells with higher maximum binding than the monospecific bivalent 5T4 antibodies IgG1-5T4-207-FEAR and IgG1-5T 4-059-FEAR. For bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (C), the maximum binding was similar on HeLa cells to that of monospecific bivalent 5T4 antibody IgG1-5T 4-226-FEAR.
FIG. 6(I) (right panel) shows that the CD3x5T4 bispecific antibody bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (A), bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (B) and bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (C) showed higher dose dependent binding to MDA-MB-231 cells than the monospecific bivalent 5T4 antibody IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T 4-059-FEAR. The negative control antibody IgG1-b12-K409R (3. mu.g/mL) contained in these experiments did not show binding to HeLa and MDA-MB-231 cells.
In a second experiment, staining was performed with minor adjustments as described above. Cells were incubated with serial dilutions of antibody ranging from 0.000128 to 10 μ g/mL in 5-fold dilution steps. As secondary antibody, Phycoerythrin (PE) -conjugated goat anti-human IgG F (ab') 2(Jackson Immunoresearch, UK, Cat. No. 109-116-098) was used at a 1:200 dilution in staining buffer.
FIGS. 6(II) and 6(III) show that the antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR, bsIgG 4-huCD 4-H101 4-FEAR, bsIgG 4-huCD 4-FEAR and IgG 4-T4-059-FEAR, bsIgG 4-huCD 4-H101 4-FEALx 5T4-106-FEAR and IgG 4-5T 4-106-FEAR, bsIgG 72-FEAR, bsIgG 4-CD 4-FEALx 5T 4-FEAR and IgG 4-FEaH 72-FEaH-4-FEaH-, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR showed dose-dependent binding to HeLa and MDA-MB-231 tumor cells. In general, compared with bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG 4-huCD 4-H101 4-FEALx 5T4-226-FEAR, bsIgG 4-CD 4-H101-FEALx 5T4-059-FEAR, bsIgG 4-FEALx 72-CD 4-H101-FEALx 5T 4-FEAR, bsIgG 4-FEALx 5T 4-FEAR, bsIgG 4-FEALx 5T 4-FEAR, FEALx 4-FEAR, FEAR 4-FEAR, FEALx, IgG1-5T4-085-FEAR and IgG1-5T4-127-FEAR showed binding at lower antibody concentrations.
Example 13-in vitro Induction by a CD3x5T4 bispecific antibody Using purified T cells as Effector cells Lead to T cell activation, cytokine release and cytotoxicity
CD3x5T4 bispecific antibodies were tested in an in vitro cytotoxicity assay using a 5T4 positive tumor cell line as target cells and purified T cells as effector cells. T cells were derived from healthy human donor buffy coats (Sanquin, Amsterdam, The Netherlands) and isolated using RosetteSep human T cell enrichment mix (Stemcell Technologies, France, cat # 15061) according to The manufacturer's instructions. To determine the percentage of live T cells after isolation (total T cells, CD 4)+T cells or CD8+T cells), isolated T cell samples (each condition 2.5x 10) were plated in U-well 96-well plates (Cellstar, catalog No. 650180) at 4 ℃ using the following antibodies5Individual cells) for 30 minutes: pacific Blue CD3(eBiosciences, clone OKT3), APC-Cy-anti-CD 4(eBiosciences, clone OKT4), AF 700-anti-CD 8(Biolegend, clone RPA-T8) and the viability marker FVS 510(BD Biosciences) in 100. mu.L PBS/0.1% BSA/0.02% azide (staining buffer). Next, the cells were washed twice in staining buffer, resuspended in 120 μ L staining buffer, and analyzed on BD LSRFortessa FACS (BD Biosciences, USA). CD3 for each donor used in the cytotoxicity experiments is described in Table 6 +,CD3+CD4+And CD3+CD8+Percentage of T cells.
Table 6: CD3 for each donor+、CD4+And CD8+T cell ratio
Figure BDA0002720035660000961
Figure BDA0002720035660000971
MDA-MB-231 cells (16,000 cells/well) were seeded into flat-bottomed 96-well plates (Greiner-bio-one, The Netherlands, Cat. 655180) and adhered at 37 ℃ for 4 hours. T cells were added to tumor cells at an E: T ratio of 8: 1. Serial dilutions of either the bispecific CD3X5T4 antibody or the monospecific bivalent 5T4 antibody (final concentration range 1000 to 0.0128 ng/mL; 5-fold dilution) were added and the plates were incubated at 37 ℃ for 72 hours. Next, 110. mu.L of the T cell-containing supernatant was transferred to a U-shaped 96-well plate (CellStar, Cat. No. 650180). Plates were centrifuged (300Xg) at 4 ℃ for 3 minutes, then 75. mu.L of supernatant was transferred to new plates for cytokine production measurements and T cells were retained to assess T cell activation markers (described below). Cytokine production induced by 0.2 μ g/mL CD3x5T4 bispecific antibody was analyzed by multiplex U-plex assay (MeSo Scale Discovery, USA, catalog No. K15049K) according to the manufacturer's instructions.
T cells were stained for the T cell markers CD3(1: 200; eBioscience, clone OKT3 conjugated to eFluor 450), CD4(1: 50; eBioscience, clone OKT4 conjugated to APC-eFluor 780), CD8(1: 100; Biolegend, clone RPA-T8 conjugated to AF 700) and the T cell activation marker CD69(1: 50; BD Biosciences, clone AB2439 conjugated to APC), CD25(1: 50; eBioscience, clone BC96 conjugated to PE-Cy 7) and CD279/PD1(1: 50; Biolegend, clone EH12.2H7 conjugated to BV 605). Single stained samples with Ultracomp beads (5. mu.L; Invitrogen, Cat. No. 01-2222-42) were used for compensatory adjustments in the flow cytometer. After incubation at 4 ℃ for 30 min, plates were washed 3 times with PBS/0.1% BSA/0.02% azide (staining buffer). Cells were resuspended in 120 μ L staining buffer and analyzed using FACS Fortessa (BD Biosciences). Data were processed using FlowJo (BD Biosciences).
In parallel, resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide)) was used to assess the viability of tumor cells. Adherent tumor cells were washed twice with PBS and incubated for 4 hours at 37 ℃ with 10% Resazurin (150. mu.L; Life Technologies, The Netherlands, Cat. No. DAL1100) in RPMI-1640(Lonza, Switzerland, Cat. No. BE12-115F) medium containing 10% iron-containing donor bovine serum (Life Technologies, The Netherlands, Cat. No. 10371-029) and penicillin/streptomycin (Lonza, Cat. No. DE 17-603E). The absorbance was measured with an Envision multi-label plate reader (PerkinElmer, US). The absorbance of the staurosporine-treated (Sigma-Aldrich, US, Cat. No. S6942) tumor cell samples was set to 0% viability and the absorbance of the untreated tumor cell samples was set to 100% viability. The "percentage of viable cells" was calculated as follows:
% living cells ═ sample absorbance-absorbance of staurosporine-treated target cells ]/[ absorbance of untreated target cells-absorbance of staurosporine-treated target cells ] x 100
Dose response curves, EC50 and IC50 values were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 7(I) shows that bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR induce dose-dependent cytotoxicity (shown as% live cell reduction) in the 5T4 positive tumor cell line MDA-MB-231. Differences between donors were observed, but the T cells of both donors induced maximal killing in the presence of 1 μ g/mL CD3x5T4 bispecific antibody. Monospecific bivalent antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not induce cytotoxicity. IC calculated from graph50The values are presented in fig. 7 (II).
IC of bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR50The values were lower compared to bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively.
In contrast, the IC of bsIgG1-huCD3-FEALx5T4-226-FEAR50The values were comparable to bsIgG1-huCD3-H101G-FEALx5T 4-226-FEAR.
FIG. 8(I) shows bsIgG-huCD-FEALx 5T-207-FEAR, bsIgG-huCD-H101-FEALx 5T-207-FEAR, bsIgG-huCD-FEALx 5T-226-FEAR, bsIgG-huCD-H101-FEALx 5T-226-FEAR, bsIgG-huCD-FEALx 5T-059-FEAR, bsIgG-huCD-H101-FEALx 5T-059-FEAR, bsIgG-huCD-FEALx 5T-106-FEAR, bsIgG-huCD-H101-FEALx 5T-A-FEAR, bsIgG1-huCD3-FEALx5T4-A3-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induced T cell mediated cytotoxicity (shown as a reduction in tumor cell survival) in the MDA-MB-231 cell line. Bivalent monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR did not induce T cell mediated cytotoxicity. The IC50 values calculated from the graph are presented in fig. 8 (II). The IC50 values of T cell mediated cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-FEALx5T4-106-FEAR were lower than the IC50 values of bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T 4-A3-FEAR. Similarly, the IC50 values of T cell-mediated cytotoxicity induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR were lower than the IC50 values of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T 4-A3-FEAR.
T cell activation was determined by flow cytometry by staining of the activation markers PD1, CD25, and CD69 (fig. 9 (I)). Monospecific bivalent antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR do not induce upregulation of these T cell activation markers, whereas bispecific antibodies bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD 1-H101 1-FEALx 5T 1-207-FEAR, bsIgG1-huCD 1-FEALx 5T 1-226-FEAR, bsIgG1-huCD 1-H101 1-FEALx 5T 1-226-FEAR, bsIgG1-huCD 1-ALx 5T 1-059-FEAR and FEbsIgG 1-CD 36101-FEALx 5T 1-FEAR, FEALx 5-059-FEAR and FEbsIgG 1-FEALx 5-FEAR do not induce upregulation of CD 1, CD 1 and CD 1-FEALx 1. EC calculated from the map50The values are presented in fig. 9 (II). bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR upregulate EC's of PD1, CD25, and CD69, compared to bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively50The value is lower. Compared with bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR up-regulates CD25 and CEC of D6950Lower value, and PD1 upregulated EC50The values were comparable between bsIgG1-huCD3-FEALx5T4-226-FEAR and bsIgG1-huCD3-H101G-FEALx5T 4-226-FEAR.
FIG. 10(I) shows bsIgG-huCD-FEALx 5T-207-FEAR, bsIgG-huCD-H101-FEALx 5T-207-FEAR, bsIgG-huCD-FEALx 5T-226-FEAR, bsIgG-huCD-H101-FEALx 5T-226-FEAR, bsIgG-huCD-FEALx 5T-059-FEAR, bsIgG-huCD-H101-FEALx 5T-059-FEAR, bsIgG-huCD-FEALx 5T-106-FEAR, bsIgG-huCD-H101-FEALx 5T-A-FEAR, bsIgG1-huCD3-FEALx5T4-A3-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induced T cell activation upon incubation with the MDA-MB-231 cell line (as CD4 in FIG. 10 (I)). +And CD8+Exemplified by an increase in% CD69+ T cells within the T cell population), whereas bivalent monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR did not induce T cell activation. EC50 values for the three T cell activation markers are shown in fig. 10 (II). Generally, EC50 values for T cell activation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-FEALx5T4-106-FEAR (CD 4)+And CD8+% CD69 within T cell population+、CD25+And PD1+Increase of cells) was lower than the EC50 values of bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T 4-A3-FEAR. Similarly, EC50 values for T cell activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR (as CD4+And CD8+% CD69 within T cell population+、CD25+And PD1+Increase in T cells) was lower than the EC50 values for bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and bsIgG1-huCD3-H101G-FEALx5T 4-A3-FEAR.
After co-culture of T cells and MDA-MB-231 cells were exposed to 0.2. mu.g/mL of CD3x5T4 bispecific antibody, cytokine IL-10, IL-13 and TNF production was measured in culture supernatants by multiplex U-plex assay. Figure 11 shows cytokine levels in the supernatant of T cell-tumor cell co-cultures after incubation with bispecific antibodies. Experiments were performed using T cells from two different healthy donors; FIG. 11A shows results from co-culture with T cells derived from donor A, and FIG. 11B shows results from co-culture with T cells derived from donor B. Although the cytokine levels in co-cultures of T-cell tumor cells incubated with CD3x5T4 bispecific antibody containing IgG1-huCD3-H101G-FEAL derived CD3 specific Fab-arm were lower than in co-cultures incubated with bispecific antibody containing IgG1-huCD3-FEAL derived CD3 specific Fab-arm, the bispecific antibody bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-H101 3-FEALx5T 3-207-FEAR, bsIgG 3-huCD 3-FEAR, bsIgG 3-huCD 3-FEx 5T 3-226-FEAR, bsIgG 3-huCD 3-H101-3-ALFEx 5-226-FEAR, bsIgG 3-CD 0572-FEX 5-FEAR and huFEX 0572-FEAR were all inducing the release of the cytokine. Monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not induce any cytokine release.
Example 14 use of PBMC or purified T cells as Effector cells, with different Effector to target ratios In vitro induction of cytotoxicity by CD3x5T4 bispecific antibody
To determine in more detail the efficiency of T cell mediated killing of the bispecific antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, cytotoxicity assays were performed as described in example 13 at different effector to target cell (E: T) ratios. In addition, Peripheral Blood Mononuclear Cells (PBMC) or isolated T cells were used as effector cells. The ovarian cancer cell line SK-OV-3(9,000 cells/well, ATCC, Cat. No. HTB-77) was used as the target cell line. PBMCs were separated from 40mL of human blood buffy coat (Sanquin) using a Ficoll gradient (Lonza; lymphocyte separation medium, Cat. No. 17-829E) according to the manufacturer's instructions. T cells were isolated as described in example 13. For PBMC, the following E: T ratios were used: 1:2,1:1,2:1,4:1,8:1 and 12: 1. For isolated T cells, the following E: T ratios were used: 1:2,1:1,2:1,4:1 and 8: 1. In each experiment, effector cells from two separate donors were used. TABLE 7 liftingCD3 in PMBC or T cell isolates supplied to each donor +,CD3+CD4+And CD3+CD8+Summary of the percentage of T cells (determined as described in example 13).
Table 7: CD3 for each donor+,CD4+And CD8+T cell ratio.
Donor % CD3 within a Living cell population CD3+Intracellular% CD4+ CD3+Intracellular% CD8+
C(PBMC) 75 56.8 28.9
D(PBMCs) 60 63.2 32
E (T cell) 98.3 59.6 31.6
F (T cell) 97.2 70 26.4
As shown in FIG. 12, using effector cells from two different donors, the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR at an E: T ratio of 4:1 to 12:1 resulted in potent PBMC-mediated killing of SK-OV-3 cells. At E: T ratios of 2:1 and lower, the maximum killing of SK-OV-3 cells was not achieved at the highest antibody concentration used (1000 ng/mL). Similar results were observed when isolated T cells were used as effector cells (fig. 13). Et ratios of 4:1 and 8:1 resulted in maximal T cell mediated killing of SK-OV-3 cells using effector cells from two different donors, at the highest antibody concentration used (1000ng/mL) in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR, whereas lower Et ratios were insufficient to induce maximal killing. Thus, the T cell mediated killing efficacy induced by bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR was dependent on a sufficiently high E: T ratio.
Example 15 anti-CD 3x5T4 bispecific antibody in humanized immune System mouse xenograft model Tumor activity
Humanization in subcutaneous inoculation with human MDA-MB-231 tumor cells (3-4 weeks old tail vein injection of CD34+ hematopoietic Stem cells [ HSC)])NOD.Cg-PrkdcscidIl2rgtm1WjlThe CD3x5T4 bispecific antibody bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR were evaluated in/SzJ (NSG-HIS) mice (obtained from The Jackson Laboratory) for in vivo anti-tumor efficacy. Humanization of the immune system of NSG-HIS mice was confirmed by flow cytometry 16 weeks after implantation. Subsequently, based on HSC donors (#5239 or #2328) and human CD45 in peripheral blood+Human CD3 in colony+Percentage of T cells (mean% hCD45, respectively)+And% hCD3+A cell; 42% hCD45 for PBS group+And 39% hCD3+For the bsIgG1-huCD3-FEALx5T4-207-FEAR group, 34% hCD45+And 25％hCD3+And 36% hCD45 for bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR+And 29% hCD3+) NSG-HIS mice were randomly divided into three groups (8 mice per group). Mix 5x106Individual MDA-MB-231 cells (in 100 μ Ι _ PBS) were injected Subcutaneously (SC) into the flank of the mice; this is indicated as day 0 in the study. Mice were injected Intravenously (IV) with 0.5mg/kg antibody or PBS on days 14, 18, 21, and 25. Treatment groups are shown in table 8. Tumor growth was assessed twice a week (from day 14) using calipers. Measured from caliper at 0.52x (length) x (width) 2Calculation of tumor volume (mm)3)。
The results are shown in fig. 14. FIG. 14A shows that, based on Mann-Whitney statistical analysis on day 43 compared to control, bsIgG1-huCD3-FEALx5T4-207-FEAR (p)<0.01) and bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p)<0.05) was effective in inhibiting tumor growth. Furthermore, statistical analysis of tumor-free survival curves using the Mantel Cox test (Kaplan Meier plot, using less than 500 mm)3Tumor as a trap) demonstrated statistically different differences in tumor-free survival, showing the use of bsIgG1-huCD3-FEALx5T4-207-FEAR (p) compared to untreated animals<0.001) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p)<0.001) increased tumor-free survival in treated animals (fig. 14B).
TABLE 8 treatment groups
Figure BDA0002720035660001031
Example 16 alanine scanning was used to determine the contribution of amino acid residue 5T4 to antibody binding.
Library design
A human 5T4(Uniprot ID Q13641) single residue alanine library (GeneArt, Thermo Fisher Scientific) was synthesized in which all amino acid residues in the human 5T4 ectodomain were individually mutated to alanine except for positions already containing alanine or cysteine. To minimize the chance of structural disruption of the antigen, no mutation is made to cysteine. The library was cloned into a pMAC expression vector containing the CMV/TK-polyA expression cassette, the ampicillin resistance gene and the pBR322 origin of replication.
Library generation and screening
Wild-type 5T4 and alanine mutants were expressed individually in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Fisher Scientific, Cat. No. 12347-019). One day after transfection, cells were harvested. Approximately 80,000 cells were incubated with 20. mu.L of FITC-conjugated antibody (3. mu.g/mL; in FACS buffer (PBS [ Lonza, Cat. BE 17-517)]+0.1％[w/v]BSA [ Roche, Cat No. 10735086001]+0.02％[w/v]Sodium azide [ NaN3(ii) a EMELCA Bioscience, catalog number 41920044-3](ii) a Table 9) incubation for 40 minutes. Subsequently, the cells were washed twice by centrifugation using 150-180. mu.L FACS buffer. Cells were resuspended in 30 μ L FACS buffer and stored at 4 ℃ until analyzed by flow cytometry using an iQue screener (Intellicyt Corporation).
The entire experiment was performed twice, yielding duplicate measurements.
Table 9: an antibody for determining the contribution of 5T4 amino acid residues in antibody binding using an alanine scan. Prior to performing the experiment, antibodies that bound univalent to 5T4 were labeled with FITC (Thermo Fisher Scientific, catalog No. 46425). IgG1-5T4-a1-F405L and IgG1-5T4-A3-F405L are surrogate a1 and A3 antibodies, respectively, cloned into the human IgG1 backbone containing the F405L mutation. Thus, the alternative a1 antibody has the same variable regions as the a1 antibody disclosed in WO 2007106744. Likewise, the A3 replacement antibody has the same variable regions as the A3 antibody disclosed in WO 2007106744. In both antibodies, the Fc domain carries the F405L substitution.
Antibodies Test or control antibodies
bsIgG1-b12-FEALx5T4-059-FEAR-FITC Test antibodies
bsIgG1-b12-FEALx5T4-207-FEAR-FITC Test antibodies
bsIgG1-b12-FEALx5T4-226-FEAR-FITC Test antibodies
bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC Test antibodies
bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC Control antibodies for normalization
Data analysis
For each sample, the average amount of antibody bound per cell was determined as the geometric mean of fluorescence intensity (gMFI) of a population of living single cells. gMFI is influenced by the affinity of the antibody for the 5T4 mutant and the expression level of the 5T4 mutant per cell. Since specific alanine mutations can affect the surface expression level of mutant 5T4, and typically correct for differences in expression for each 5T4 mutant, the data for each test antibody was normalized to the binding strength of a non-cross-blocking 5T 4-specific control antibody using the following equation:
Figure BDA0002720035660001051
wherein "aa position" refers to a position mutated to alanine and a Z score is calculated to express the loss or gain of binding of the antibody according to the following equation:
Figure BDA0002720035660001052
where μ and σ are the mean and standard deviation, respectively, of the normalized gMFI calculated from all mutants,
wherein data were excluded from the analysis if the gMFI of the control antibody for the particular 5T4 mutant was lower than the SD of the mean gMFI control antibody-2.5 × mean gMFI control antibody (from all mutants) (assuming that the expression level of those 5T4 mutants was not sufficient to draw conclusions). This is the case for amino acid W at position 296(SEQ ID NO: 1).
Results
Figure 15 shows the antibodies tested in combination with the ECD: binding results for the human 5T4 variant with a single alanine mutation in positions 32 to 355 (according to SEQ ID NO: 1). The results indicate that antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC shows a loss of binding when aa R at position 73, T at position 74, Y at position 92, R at position 94, N at position 95 or F at position 138 of human 5T4 is mutated to alanine. This suggests that binding of antibody IgG1-5T4-059-04-FEAR is dependent on at least R73, T74, Y92, R94, N95, F138 of human 5T4(SEQ ID NO:1), and antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC shows loss of binding when aa S at position 69, R at position 73, Y at position 92, R at position 94, F at position 111, F at position 138, D at position 148 of human 5T4 are mutated to alanine. This suggests that binding of antibody IgG1-5T4-207-FEAR is dependent on at least aa S69, R73, Y92, R94, F111, F138, and D148 of human 5T4(SEQ ID NO:1), and that antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC shows a loss of binding when aa R at position 73, Y at position 92, R at position 94, F at position 111, F at position 138, L at position 144, or D at position 148 of human 5T4 is mutated to alanine. This suggests that binding of antibody IgG1-5T4-226-FEAR is dependent on at least aa R73, Y92, R94, F111, F138, L144, and D148 of human 5T4(SEQ ID NO:1), and that antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC shows binding loss when aa D at position 60, Q at position 61, D at position 88, L at position 89, Y at position 92, F at position 111, P at position 115, L at position 117, F at position 138, D at position 148, or N at position 152 of human 5T4 is mutated to alanine. This suggests that binding of antibody IgG1-5T4-A3-FEAR is dependent on at least aa D60, Q61, D88, L89, Y92, F111, P115, L117, F138, D148 and N152 of human 5T4(SEQ ID NO: 1).
Some amino acids may be involved indirectly in binding. For example, mutation of a hydrophobic residue to alanine can affect local folding and affect the positioning of residues that interact directly (Zhao et al, 2014Structure 22, 612-620). According to the structural data (human 5T4 crystal structure 4 cnm; RCSB protein database), the following residues are buried and thus are expected to contribute indirectly to the binding:
antibody bsIgG1-b12-FEALx5T4-059-04-FEAR-FITC F138,
antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC F111, F138, D148,
antibodies bsIgG1-b12-FEALx5T4-226-FEAR-FITC F111, F138, L144, D148,
antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC L89, F111, L117, F138, D148, N152.
Since only surface exposed residues can interact directly with the antibody, the following residues are expected to interact directly with the following antibodies:
antibodies bsIgG1-b12-FEALx5T4-059-FEAR-FITC R73, T74, Y92, R94 and N95,
antibodies bsIgG1-b12-FEALx5T4-207-FEAR-FITC S69, R73, Y92 and R94,
antibodies bsIgG1-b12-FEALx5T4-226-FEAR-FITC R73, Y92 and R94,
antibodies bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC D60, Q61, D88, Y92 and P115.
Together, these results suggest that antibodies IgG1-5T4-059, IgG1-5T4-207, and IgG1-5T4-226 all bind by direct interaction with amino acid residues R73, Y92, and R94. The results also indicate that antibodies IgG1-5T4-059, IgG1-5T4-207, and IgG1-5T4-226 each bind an epitope that is different from, but partially overlapping with, the epitope bound by IgG1-5T 4-A3. This is consistent with the permutation behaviour described in examples 3 and 4.
Example 17: CD3x5T4 bispecific antibodies induce T cell activation and cytotoxicity in vitro in cell lines of different indications
The CD3x5T4 bispecific antibody was tested in an in vitro cytotoxicity assay using tumor cell lines of pancreatic and cervical cancer as target cells and purified T cells as effector cells. For each indication (pancreatic and cervical cancer), two representative cell lines were selected. Table 10 summarizes the tumor cell lines used in the in vitro cytotoxicity assay. T cells were derived from human donor buffy coat (Sanquin, Amsterdam, The Netherlands) and isolated using RosetteSep human T cell enrichment mixture (Stemcell Technologies, France, cat. No. 15061) according to The manufacturer's instructions. For each cell line, at least three different donors were tested in an in vitro cytotoxicity assay and T cell activation assay, as summarized in table 10.
Table 10: tumor cell lines for in vitro cytotoxicity assays
Figure BDA0002720035660001071
Tumor cells (16,000 cells/well) were seeded into flat-bottomed 96-well plates (Greiner Bio-One, The Netherlands, Cat. 655180) and adhered at 37 ℃ for 4 hours. T cells were treated with E: t ratio 4:1 was added to tumor cells. Serial dilutions (final concentration range 5000 to 0.0128 ng/mL; 5-fold dilution) of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibody (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) were added and the plates were incubated at 37 ℃ for 72 hours. Next, 110. mu.L of the T cell-containing supernatant was transferred to a round-bottom 96-well plate (CellStar, Cat. No. 650180) and centrifuged (300Xg) at 4 ℃ for 3 minutes. T cells were stained for the T cell markers by incubation with CD3-eFluor450 (1: 200; eBioscience, clone OKT3), CD4-APC-eFluor780(1: 50; eBioscience, clone OKT4), CD8-AF700(1: 100; Biolegend, clone RPA-T8) and the T cell activation markers CD69-APC (1: 50; Biosciences, clone AB2439), CD25-PE-Cy7(1: 50; eBioscience, clone BC96) and CD279/PD1-BV605(1: 50; Biolegend, clone EH12.2H7) diluted in 50. mu.L PBS/0.1% BSA/0.02% azide (staining buffer). Single stained samples with Ultracomp beads (5. mu.L; Invitrogen, Cat. No. 01-2222-42) were used for compensatory adjustments in the flow cytometer. After incubation at 4 ℃ for 30 min, the plates were washed 3 times with staining buffer. Cells were resuspended in 120 μ L staining buffer and analyzed using FACS Fortessa (BD Biosciences). Data were processed using FlowJo (10 th edition, BD Biosciences).
In parallel, resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) was used to assess the viability of tumor cells. Adherent tumor cells were washed twice with PBS and incubated for 4 hours at 37 ℃ with 10% resazurin (150. mu.L; Life Technologies, The Netherlands, Cat. No. 10371-029) in RPMI-1640 medium (Lonza, Switzerland, Cat. No. BE12-115F) supplemented with 10% iron-containing donor bovine serum (Life Technologies, The Netherlands, Cat. No. 10371-029) and penicillin/streptomycin (Lonza, Cat. No. DE 17-603E). The absorbance was measured with an Envision multi-label plate reader (PerkinElmer, US). The absorbance of staurosporine-treated (Sigma-Aldrich, US, Cat. No. S6942) cells was set to 0% viability and the absorbance of untreated cells was set to 100% viability. The "percentage of viable cells" was calculated as follows:
% living cells ═ sample absorbance-absorbance of staurosporine-treated target cells ]/[ absorbance of untreated target cells-absorbance of staurosporine-treated target cells ] x 100
Dose cytotoxicity curves, T cell activation curves, IC50 (cytotoxicity) and EC50(T cell activation) values were analyzed using nonlinear regression (sigmoidal dose response with variable slope) using GraphPad Prism V7.02 Software (GraphPad Software, San Diego, CA, USA).
FIG. 16(I) shows that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR induced cytotoxicity in a panel of cell lines for different indications, whereas control bispecific antibodies targeting only tumor cells or T cells (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) did not show any cytotoxicity. Figure 16(II) shows the mean IC of each cell line tested using different donors (at least n-3)50The value is obtained. FIG. 17(I) shows that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR induces T cell activation in a panel of different indicated cell lines, as by CD4+And CD8+CD69 on T cells (CD 4)+Or CD8+Intra-population CD69+Cell%) was measured. Control bispecific antibodies targeting only tumor cells or T cells (bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR) did not induce any T cell activation. Figure 17(II) shows the mean EC for each cell line tested using different donors (at least n-3)50The value is obtained.
These data indicate that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR can specifically induce T cell mediated cytotoxicity and T cell activation in pancreatic and cervical cancers, while control bispecific antibodies bsIgG1-huCD3-H101G-FEALxb12-FEAR and bsIgG1-b12-FEALx5T4-207-FEAR do not induce T cell activation and T cell mediated cytotoxicity.
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72.Van Heeke&Schuster,J Biol Chem 264,5503 5509(1989)
73.Remington:The Science and Practice of Pharmacy,19th Edition,Gennaro,Ed.,Mack Publishing Co.,Easton,PA,1995
74.Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978
75.US 4,699,880
76.Kozak,M.,Gene 1999；234(2):187-208)
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79.Lefranc MP.et al.,Nucleic Acids Research,27,209-212,1999
80.Brochet X.Nucl.Acids Res.36,W503-508(2008)
81.WO2007/106744
82.WO2015/001085
83.WO2017/009442
84.WO2011147986,
85.WO2011131746
86.WO2013060867
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90.WO2007/106744
91.US 2015/0337049
Sequence listing
<110> Jianma Bao
Satijn, David
Parren, Paul
Horbach, Sjeng
Verzijl, Dennis
Rademakers, Rik
van den Brink, Edward
Engelberts, Patrick
Kemper, Kristel
de Goeij, Bart
Breij, Esther
<120> antibody
<130> P/0131-WO-PCT
<150> EP18161293.8
<151> 2018-03-12
<150> EP 18175347.6
<151> 2018-05-31
<160> 105
<170> PatentIn version 3.5
<210> 1
<211> 420
<212> PRT
<213> Intelligent people
<400> 1
Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu
1 5 10 15
Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser
20 25 30
Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu
35 40 45
Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala
50 55 60
Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg
65 70 75 80
Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu
85 90 95
Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala
100 105 110
Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser
115 120 125
Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu
130 135 140
Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe
145 150 155 160
Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val
165 170 175
Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg Gln Asn
180 185 190
Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala
195 200 205
Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr
210 215 220
Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp
225 230 235 240
Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn
245 250 255
Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val
260 265 270
Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg
275 280 285
Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp
290 295 300
Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Asp Arg
305 310 315 320
Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu
325 330 335
Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu
340 345 350
Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala
355 360 365
Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp
370 375 380
Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His
385 390 395 400
Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser
405 410 415
Asn Ser Asp Val
420
<210> 2
<211> 420
<212> PRT
<213> monkey in bunches
<400> 2
Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu
1 5 10 15
Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser
20 25 30
Ser Ser Thr Ser Ser Ala Ser Ser Ser Ser Ser Ser Ala Pro Phe Leu
35 40 45
Ala Ser Ala Ala Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala
50 55 60
Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg
65 70 75 80
Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Leu Tyr Val Arg Asn Leu
85 90 95
Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala
100 105 110
Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser
115 120 125
Arg Leu Asp Glu Val Arg Gly Gly Ala Phe Glu His Leu Pro Ser Leu
130 135 140
Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Tyr Leu Ser Pro Phe
145 150 155 160
Ala Phe Ser Gly Ser Asn Ala Ser Ile Ser Ala Pro Ser Pro Leu Val
165 170 175
Glu Leu Ile Leu Asn His Ile Val Pro Pro Asp Asp Lys Arg Gln Asn
180 185 190
Arg Ser Phe Glu Gly Met Val Ala Ala Ala Leu Val Ala Gly Arg Ala
195 200 205
Leu Gln Gly Leu His Leu Leu Glu Leu Ala Ser Asn His Phe Leu Tyr
210 215 220
Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg Tyr Leu Asp
225 230 235 240
Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn
245 250 255
Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val
260 265 270
Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Val Arg
275 280 285
Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp
290 295 300
Met Val Thr Trp Leu Lys Gln Thr Gly Val Val Gln Gly Lys Asp Arg
305 310 315 320
Leu Thr Cys Ala Phe Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu
325 330 335
Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu
340 345 350
Gln Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala
355 360 365
Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp
370 375 380
Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr His
385 390 395 400
Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser
405 410 415
Asn Ser Asp Val
420
<210> 3
<211> 379
<212> PRT
<213> original chicken
<400> 3
Met Pro Gly Arg Glu Ala Glu Arg Arg Gly Ala Leu Cys Leu Gly Leu
1 5 10 15
Leu Leu His Ala Leu Leu Gly Cys Gly Ser Ala Gln Pro Pro Ala Ala
20 25 30
Cys Pro Ala Pro Cys Glu Cys Ser Glu Ala Ala Lys Thr Val Lys Cys
35 40 45
Val Asn Lys Asn Leu Thr Glu Val Pro Pro Asp Leu Pro Pro Tyr Val
50 55 60
Arg Asn Leu Phe Ile Thr Gly Asn Arg Leu Gly Arg Leu Pro Ala Gly
65 70 75 80
Ala Leu Ser Ala Pro Arg Leu Ala Glu Leu Gly Ser Leu Asn Leu Ser
85 90 95
Gly Asn His Leu Arg Ala Val Glu Ala Gly Ala Leu Ala Ala Leu Pro
100 105 110
Ala Leu Arg Gln Leu Asp Leu Gly Gly Asn Pro Leu Ala Glu Leu Ser
115 120 125
Pro Leu Ala Phe Gly Arg Ala Ser Pro Leu Glu Glu Leu Ala Leu Arg
130 135 140
Gly Ala Leu Arg Glu Gln Gly Ala Leu Leu Gly Leu Ala Asp Leu Leu
145 150 155 160
Gln Ala Gly Ala Leu Arg Asn Leu Ser Arg Leu Glu Leu Ala Asp Asn
165 170 175
Gly Leu Leu Leu Leu Pro Thr Gly Met Leu Gly Ala Leu Pro Ala Leu
180 185 190
Arg His Leu Asp Leu Ser Asn Asn Ser Leu Val Gly Leu Arg Asn Val
195 200 205
Ser Phe Gln Gly Leu Val Arg Leu Gln Ser Leu Asn Leu Ser Asp Asn
210 215 220
Ser Leu Gly Val Leu Arg Asn Gly Thr Leu Ala Gln Trp Arg Gly Leu
225 230 235 240
Pro Ala Leu Arg Arg Ile Ser Leu Ser His Asn Thr Trp Val Cys Asp
245 250 255
Cys Ala Ile Glu Asp Met Val Ala Trp Leu Lys Glu Ser Asp Gln Val
260 265 270
Glu Gly Lys Glu Ala Leu Ser Cys Ala Phe Pro Glu Lys Met Ala Gly
275 280 285
Arg Ala Leu Leu Lys Leu Asn Thr Ser Glu Leu Asn Cys Ser Ala Pro
290 295 300
Val Asp Val Pro Ser Gln Leu Gln Thr Ser Tyr Val Phe Leu Gly Ile
305 310 315 320
Val Leu Ala Leu Ile Gly Ala Ile Phe Leu Leu Val Leu Tyr Leu Asn
325 330 335
Arg Lys Gly Ile Lys Lys Trp Met His Asn Ile Arg Asp Ala Cys Arg
340 345 350
Asp His Met Glu Gly Tyr His Tyr Arg Tyr Glu Ile Asn Ala Asp Pro
355 360 365
Arg Leu Thr Asn Leu Ser Ser Asn Ser Asp Val
370 375
<210> 4
<211> 186
<212> PRT
<213> Intelligent people
<400> 4
Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys
1 5 10 15
Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro
20 25 30
Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp
35 40 45
Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys
50 55 60
Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg
65 70 75 80
Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg
85 90 95
Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile
100 105 110
Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr
115 120 125
Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly
130 135 140
Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro
145 150 155 160
Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp
165 170 175
Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile
180 185
<210> 5
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable domains
<400> 5
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Thr Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 6
Gly Phe Thr Phe Ser Ser Tyr Asp
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 7
Ile Ser Tyr Asp Gly Ser Asn Lys
1 5
<210> 8
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 8
Ala Arg Asp Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met
1 5 10 15
Asp Val
<210> 9
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable domains
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 10
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 10
Gln Gly Ile Ser Ser Trp
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 11
Gln Gln Tyr Asn Ser Tyr Pro Leu Thr
1 5
<210> 12
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable domains
<400> 12
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60
Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Arg Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Gly Tyr Phe Asp Trp Leu Tyr Gly Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 13
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 13
Gly Tyr Thr Phe Thr Ser Tyr Gly
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 14
Ile Ser Ala Tyr Asn Gly Asn Thr
1 5
<210> 15
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 15
Ala Arg Asp Pro Gly Tyr Phe Asp Trp Leu Tyr Gly Asp Tyr
1 5 10
<210> 16
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 16
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Arg
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 17
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 17
Gln Gly Ile Ser Ser Ala
1 5
<210> 18
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 18
Gln Gln Phe Asn Ser Tyr Pro Arg Thr
1 5
<210> 19
<211> 121
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 19
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Asn Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu His Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Pro Gly Tyr Asn Asn Val Glu Tyr Leu Asp His Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 20
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 20
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 21
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 21
Ile Ser Gly Ser Gly Gly Ser Thr
1 5
<210> 22
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 22
Ala Arg Asp Pro Gly Tyr Asn Asn Val Glu Tyr Leu Asp His
1 5 10
<210> 23
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 23
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 24
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 24
Gln Gly Ile Ser Ser Ala
1 5
<210> 25
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 25
Gln Gln Phe Asn Ser Tyr Pro Leu Thr
1 5
<210> 26
<211> 115
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 26
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Arg Phe Thr Ser Tyr
20 25 30
Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ala Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Gly Met Tyr Tyr Cys
85 90 95
Ala Arg Ser Val Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ser
115
<210> 27
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 27
Gly Tyr Arg Phe Thr Ser Tyr Trp
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 28
Ile Tyr Pro Gly Asp Ser Asp Ala
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 29
Ala Arg Ser Val Leu Phe Asp Tyr
1 5
<210> 30
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 30
Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Asp Val Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro His
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 31
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 31
Gln Gly Ile Ser Ser Ala
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 32
Gln Gln Phe Asn Ser Tyr Pro His Thr
1 5
<210> 33
<211> 123
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 33
Glu Val Gln Leu Leu Glu Ser Arg Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 34
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 34
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 35
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 35
Ile Ser Gly Ser Gly Gly Ser Thr
1 5
<210> 36
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 36
Ala Lys Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His
1 5 10 15
<210> 37
<211> 108
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 37
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Leu Met
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 38
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 38
Gln Ser Val Ser Ser Tyr
1 5
<210> 39
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 39
Gln Gln Arg Ser Asn Trp Leu Met Tyr Thr
1 5 10
<210> 40
<211> 122
<212> PRT
<213> Artificial sequence
<400> 40
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asp His Ser Glu Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Gly Trp Phe Gly Glu Leu Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 41
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 41
Gly Gly Ser Phe Ser Gly Tyr Tyr
1 5
<210> 42
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 42
Ile Asp His Ser Glu Ser Thr
1 5
<210> 43
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 43
Ala Gly Trp Phe Gly Glu Leu Tyr His Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210> 44
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 44
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 45
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 45
Gln Ser Val Ser Ser Tyr
1 5
<210> 46
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 46
Gln Gln Arg Ser Asn Trp Pro Leu Thr
1 5
<210> 47
<211> 122
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 47
Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Trp Phe Gly Glu Leu Trp Asp Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 48
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 48
Gly Gly Ser Phe Ser Gly Tyr Tyr
1 5
<210> 49
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 49
Ile Asp His Ser Gly Ser Thr
1 5
<210> 50
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 50
Ala Ala Trp Phe Gly Glu Leu Trp Asp Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210> 51
<211> 107
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 51
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Phe
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 52
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 52
Gln Ser Val Ser Ser Phe
1 5
<210> 53
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 53
Gln Gln Arg Ser Asn Trp Pro Leu Thr
1 5
<210> 54
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 54
Gly Phe Thr Phe Asn Thr Tyr Ala
1 5
<210> 55
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> CDr sequence
<400> 55
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr
1 5 10
<210> 56
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 56
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
1 5 10 15
<210> 57
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 57
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 58
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 58
Thr Gly Ala Val Thr Thr Ser Asn Tyr
1 5
<210> 59
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> CDR sequences
<400> 59
Ala Leu Trp Tyr Ser Asn Leu Trp Val
1 5
<210> 60
<211> 109
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 60
Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser
20 25 30
Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly
35 40 45
Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala
65 70 75 80
Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn
85 90 95
Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 61
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 61
Gly Phe Thr Phe Asn Pro Tyr Ala
1 5
<210> 62
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 62
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Pro Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 63
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 63
Gly Phe Thr Phe Asn Met Tyr Ala
1 5
<210> 64
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 64
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 65
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 65
Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr
1 5 10
<210> 66
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> antibody variable region
<400> 66
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 67
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 67
Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
1 5 10 15
<210> 68
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 68
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 69
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 69
Val Arg Asn Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
1 5 10 15
<210> 70
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 70
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg Asn Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 71
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 71
Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe Ala Tyr
1 5 10 15
<210> 72
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 72
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 73
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 73
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ala Trp Phe Ala Tyr
1 5 10 15
<210> 74
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 74
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ala Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 75
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 75
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Gly Trp Phe Ala Tyr
1 5 10 15
<210> 76
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 76
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Gly Trp Phe
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 77
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 77
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Val
1 5 10 15
<210> 78
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 78
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 79
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 79
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Met
1 5 10 15
<210> 80
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 80
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Met Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 81
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 81
Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Arg
1 5 10 15
<210> 82
<211> 125
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 82
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
100 105 110
Ala Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 83
<211> 119
<212> PRT
<213> Intelligent people
<400> 83
Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu
1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe
20 25 30
Gly Met Asn Trp Val Lys Gln Gly Pro Gly Glu Gly Leu Lys Trp Met
35 40 45
Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Arg Tyr Ala Glu Glu Phe
50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Thr Ala Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 84
<211> 107
<212> PRT
<213> Intelligent people
<400> 84
Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Asn Phe Ala Thr Asn Arg Tyr Thr Gly Val Pro Asn Arg Phe Thr Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Leu Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 85
<211> 122
<212> PRT
<213> Intelligent people
<400> 85
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met
65 70 75 80
Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95
Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 86
<211> 107
<212> PRT
<213> Intelligent people
<400> 86
Asp Ile Val Met Thr Gln Ser His Ile Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 87
<211> 120
<212> PRT
<213> Intelligent people
<400> 87
Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Asn Asn Gly Val Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met Asp Tyr Trp Gly Gln
100 105 110
Val Thr Ser Val Thr Val Ser Ser
115 120
<210> 88
<211> 107
<212> PRT
<213> Intelligent people
<400> 88
Ser Ile Val Met Thr Gln Thr Pro Thr Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile
35 40 45
Ser Tyr Thr Ser Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly
50 55 60
Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 89
<211> 330
<212> PRT
<213> Intelligent people
<400> 89
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 90
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 90
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 91
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 91
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 92
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 92
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 93
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 93
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 94
<211> 330
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 94
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 95
<211> 107
<212> PRT
<213> Intelligent people
<400> 95
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 96
<211> 106
<212> PRT
<213> Intelligent people
<400> 96
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 97
<211> 127
<212> PRT
<213> Intelligent people
<400> 97
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe
20 25 30
Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe
50 55 60
Gln Asp Arg Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr
100 105 110
Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser
115 120 125
<210> 98
<211> 108
<212> PRT
<213> Intelligent people
<400> 98
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg Ser Arg
20 25 30
Arg Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val
35 40 45
Ile His Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu
65 70 75 80
Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly Ala Ser Ser
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys
100 105
<210> 99
<211> 363
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 99
Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu
1 5 10 15
Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser
20 25 30
Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu
35 40 45
Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala
50 55 60
Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg
65 70 75 80
Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu
85 90 95
Phe Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala
100 105 110
Arg Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser
115 120 125
Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu
130 135 140
Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe
145 150 155 160
Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val
165 170 175
Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg Gln Asn
180 185 190
Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala
195 200 205
Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr
210 215 220
Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp
225 230 235 240
Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn
245 250 255
Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val
260 265 270
Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg
275 280 285
Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp
290 295 300
Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Asp Arg
305 310 315 320
Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu
325 330 335
Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu
340 345 350
Gln Thr Ser His His His His His His His His
355 360
<210> 100
<211> 327
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 100
Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu
1 5 10 15
Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser
20 25 30
Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe Leu
35 40 45
Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys Pro Ala
50 55 60
Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg
65 70 75 80
Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala Ala Pro Ser Thr Cys
85 90 95
Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val
100 105 110
Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
115 120 125
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu
130 135 140
Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg
145 150 155 160
Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser
165 170 175
Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys
180 185 190
Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
195 200 205
Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly
210 215 220
Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met
225 230 235 240
Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn
245 250 255
Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser
260 265 270
Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu
275 280 285
Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu
290 295 300
His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys His
305 310 315 320
His His His His His His His
325
<210> 101
<211> 276
<212> PRT
<213> Artificial sequence
<220>
<223> N/A
<400> 101
Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp
1 5 10 15
Pro Met Val Trp Ala Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr
20 25 30
Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr
35 40 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
50 55 60
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu
65 70 75 80
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
85 90 95
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
100 105 110
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser
115 120 125
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
130 135 140
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile Thr
145 150 155 160
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
165 170 175
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
180 185 190
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
195 200 205
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
210 215 220
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
225 230 235 240
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
245 250 255
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
260 265 270
Asn Arg Gly Glu
275
<210> 102
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR consensus sequence
<400> 102
Tyr Tyr Gly Met Asp Val
1 5
<210> 103
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> CDR consensus sequence
<220>
<221> Xaa
<222> (5)..(5)
<223> Xaa is G or E
<400> 103
Ile Asp His Ser Xaa Ser Thr
1 5
<210> 104
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> CDR consensus sequence
<220>
<221> Xaa
<222> (2)..(2)
<223> Xaa is A or G
<220>
<221> Xaa
<222> (8)..(8)
<223> Xaa is W or Y
<220>
<221> Xaa
<222> (9)..(9)
<223> Xaa is D or H
<400> 104
Ala Xaa Trp Phe Gly Glu Leu Xaa Xaa Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210> 105
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> CDR consensus sequence
<220>
<221> Xaa
<222> (6)..(6)
<223> Xaa is Y or F
<400> 105
Gln Ser Val Ser Ser Xaa
1 5

Claims (133)

1. An antibody comprising at least one antigen binding region capable of binding 5T4 (trophoblast glycoprotein), wherein the antibody is capable of blocking the binding of an antibody [059] comprising a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID NO:5 and a light chain Variable (VL) region comprising the sequence set forth in SEQ ID NO:9 to 5T 4.
2. The antibody of claim 1, wherein the antibody blocks binding to 5T4 of an antibody selected from the group consisting of:
a) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:40 and a light chain Variable (VL) region [207] comprising the sequence shown as SEQ ID NO:44,
b) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:47 and a light chain Variable (VL) region [226] comprising the sequence shown as SEQ ID NO: 51; and
c) an antibody comprising a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID NO. 5 and a light chain Variable (VL) region comprising the sequence set forth in SEQ ID NO. 9 [059 ].
3. The antibody of claim 1 or 2, wherein the antibody blocks binding to 5T4 of an antibody selected from the group consisting of:
a) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:40 and a light chain Variable (VL) region [207] comprising the sequence shown as SEQ ID NO: 44; and
b) an antibody comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:47 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO:51 [226 ].
4. The antibody according to any one of the preceding claims, wherein 5T4 is human (homo sapiens) 5T4 as the mature polypeptide sequence of SEQ ID NO: 1.
5. The antibody according to any one of claims 1 to 3, wherein 5T4 is the mature polypeptide sequence of cynomolgus monkey (cynomolgus monkey) 5T4 as set forth in SEQ ID NO 2.
6. The antibody according to any one of claims 1-3, wherein 5T4 is chicken (gallus domesticus) 5T4 as the mature polypeptide sequence of SEQ ID NO 3.
7. The antibody according to any one of the preceding claims, wherein 5T4 is human 5T4, mature polypeptide as SEQ ID NO:1, and cynomolgus monkey 5T4, mature polypeptide as SEQ ID NO: 2.
8. The antibody according to any one of the preceding claims, wherein 5T4 is human 5T4, mature polypeptide sequence as set forth in SEQ ID NO 1, cynomolgus monkey 5T4, mature polypeptide sequence as set forth in SEQ ID NO 2, and chicken 5T4, mature polypeptide sequence as set forth in SEQ ID NO 3.
9. The antibody according to any one of the preceding claims, which is capable of corresponding to KDA value of 1E-7M or less, such as 5E-8M or less, 1E-8M or less, 5E-9M or less, or such as 1E-9M or less, binding affinity, such as in a range corresponding to 1E-7 to 5E-10M, such as 1E-7 to 1E-9M,e.g., 5E-8 to 5E-10M, 5E-8 to 1E-9M, e.g., 1E-8 to 5E-10M, 1E-8 to 1E-9M or K in the range of 1E-8 to 5E-9MDThe binding affinities of values bind to human 5T4, cynomolgus monkey and/or chicken 5T 4.
10. The antibody according to claim 9, wherein the binding affinity is determined by biolayer interferometry, optionally as described in example 2 herein.
11. The antibody according to any one of claims 9 and 10, wherein the binding affinity is determined using a biolayer interferometry comprising the steps of:
I) an amount of 1. mu.g/mL of the antibody was immobilized on an anti-human IgG Fc capture biosensor for 600 seconds;
II) 5T4ECDHis (mature protein of SEQ ID NO: 99) or cynomolgus monkey 5T4 (mature protein of SEQ ID NO: 2) or recombinant cynomolgus monkey 5T4 protein (Cusabio; directory number CSB-MP024093MOV) association over a period of 200 seconds and dissociation over a period of 1000 seconds,
III) reference data against buffer control (0 nM).
12. The antibody according to any one of claims 9-11, wherein the binding affinity is determined using an antibody as defined in any one of the preceding claims, which is a monospecific bivalent antibody, such as an antibody which is a full length IgG 1.
13. The antibody according to any one of the preceding claims, wherein said antibody recognizes an epitope on 5T4 or an antibody binding region or binding site, which is recognized by any one of the antibodies selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 5 and a VL region comprising the sequence shown as SEQ ID NO. 9 [059],
b) An antibody comprising a VH region comprising the sequence shown as SEQ ID NO 12 and a VL region [076] comprising the sequence shown as SEQ ID NO 16,
c) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 19 and a VL region [085] comprising the sequence shown as SEQ ID NO 23,
d) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 26 and a VL region [106] comprising the sequence shown as SEQ ID NO 30,
e) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 33 and a VL region [127] comprising the sequence shown as SEQ ID NO 37,
f) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO 40 and a VL region [207] comprising the sequence shown as SEQ ID NO 44; and
g) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO. 47 and a VL region comprising the sequence shown as SEQ ID NO. 51 [226 ].
14. The antibody according to any one of the preceding claims, wherein the antibody binds to an antibody binding region, binding site or epitope on 5T4 that is not an antibody binding region or binding site or epitope bound by or different from an antibody binding region, binding site or epitope bound by an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO:87 and a VL region comprising the sequence shown as SEQ ID NO:88 [ H8],
b) An antibody comprising a VH region comprising the sequence shown as SEQ ID NO:83 and a VL region comprising the sequence shown as SEQ ID NO:84 [ A1 ]; and
c) an antibody comprising a VH region comprising the sequence shown as SEQ ID NO:85 and a VL region comprising the sequence shown as SEQ ID NO:86 [ A3 ].
15. The antibody according to any one of the preceding claims, wherein binding of the antibody to 5T4 is blocked by binding of 5T4 to an antibody [ A3] comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:85 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO: 86.
16. The antibody according to any one of the preceding claims, wherein the antibody shows a substitution of an antibody binding to 5T4ECDHis (mature protein of SEQ ID NO: 99), said antibody binding to 5T4 comprising a heavy chain Variable (VH) region comprising the sequence shown in SEQ ID NO:85 and a light chain Variable (VL) region [ A3] comprising the sequence shown in SEQ ID NO: 86.
17. The antibody according to claim 14 or 15, wherein the ability of the cross-blocking or antibody as defined in any of the preceding claims to block binding of another antibody to 5T4 is determined by a Fluorescence Activated Cell Sorting (FACS) assay, e.g. an assay performed as described in example 5.
18. The antibody according to any one of claims 14 to 16, wherein the ability of a cross-blocking or an antibody as defined in any one of the preceding claims to block binding of another antibody to 5T4 is determined as the ability of an unconjugated antibody to block binding of a conjugated antibody and is optionally determined in a procedure comprising the steps of:
i) providing a set of samples, each sample comprising a mixture of human ovarian adenocarcinoma SK-OV-3 cells, an antibody that binds 5T4 and is conjugated to Fluorescein Isothiocyanate (FITC), and an excess of unconjugated antibody that targets 5T4,
ii) incubating the sample at 4 ℃ for 30 minutes and then centrifuging the sample,
iii) removing the supernatant from each sample and resuspending the cells in buffer and determining the Mean Fluorescence Intensity (MFI) of FITC using a flow cytometer; and is
iv) percent binding was calculated as follows:
the difference in MFI between cells incubated with the mixture of FITC-conjugated and unconjugated antibodies and cells not incubated with FITC-conjugated or unconjugated antibodies was multiplied by 100 and then divided by the difference in MFI between cells incubated with the mixture of FITC-conjugated and IgG-b12 antibodies and cells not incubated with FITC-conjugated or unconjugated antibodies.
19. The antibody according to any one of claims 14 to 17, wherein the ability of the antibody to block binding of another antibody to 5T4 or to displace another antibody that binds to 5T4ECDHis (mature protein of SEQ ID NO: 99) is determined using biolayer interferometry, for example as described in example 3.
20. The antibody according to any one of claims 14 to 18, wherein the ability of the antibody to block binding of another antibody to 5T4 or displace another antibody that binds to 5T4 is determined using biolayer interferometry in a procedure comprising the steps of:
i) immobilizing an antibody according to any one of the preceding claims to the activated amine-reactive second-generation biosensor in an amount of 20 μ g/mL in 10mM sodium acetate buffer,
ii) quenching the biosensor with immobilized antibody in ethanolamine at pH 8.5,
iii) the biosensor with immobilized antibody was immersed in a composition comprising 3.6. mu.g/mL (100nM) of human 5T4ECDHI (mature protein of SEQ ID NO: 99) for a period of 500 seconds, followed by
iv) immersing the biosensor with immobilized antibody and 5T4ECDHis in a composition comprising 10 μ g/mL of an additional antibody targeting 5T4 and determining the association response over a period of 500 seconds;
Wherein steps i) -iv) are performed at a temperature of 30 ℃ and with shaking at 1000 rpm.
21. The antibody according to any one of the preceding claims, wherein the antibody binds to an epitope or antibody binding region on human 5T4 comprising amino acid residues R73, Y92, and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
22. The antibody according to any one of the preceding claims, wherein the antibody binds to an epitope or antibody binding region on human 5T4 comprising amino acid residues S69, R73, Y92 and R94; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
23. The antibody according to any one of claims 1-21, wherein the antibody binds to an epitope or antibody binding region on human 5T4 comprising amino acid residues R73, T74, Y92, R94 and N95; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
24. An antibody according to any one of claims 21 to 23 wherein the amino acid residues are directly involved in the binding of the antibody.
25. The antibody according to any one of claims 21 to 24, wherein one or more of the following further amino acid residues are involved in the binding of the antibody, such as being indirectly involved in binding, for example by affecting the protein folding and/or localization of one or more amino acid residues directly involved in the binding of the antibody: l89, F111, L117, F138, L144, D148, N152; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
26. The antibody according to any one of the preceding claims, wherein the antibody binds to an epitope on human 5T4 in which amino acid residues R73, Y92 and R94 are directly involved in binding the antibody, or an antibody binding region in which one or more of amino acid residues F111, F138, L144 and D148 are indirectly involved in the binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
27. The antibody according to any one of claims 1 to 25, wherein the antibody binds to an epitope on human 5T4 in which amino acid residues S69, R73, Y92 and R94 are directly involved in binding the antibody, or an antibody binding region in which one or more of amino acid residues F111, F138 and D148 are indirectly involved in the binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
28. The antibody according to any one of claims 1 to 25, wherein the antibody binds to an epitope on human 5T4 in which amino acid residues R73, T74, Y92, R94 and N95 are directly involved in binding to the antibody, or an antibody binding region in which amino acid residue F138 is indirectly involved in the binding; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
29. The antibody according to any one of claims 21 to 28, wherein said amino acid residues comprised by said epitope or antibody binding region and optionally said one or more further amino acid residues are identified by alanine scanning of human 5T4 having the amino acid sequence shown in SEQ ID No. 1 or the mature polypeptide sequence of SEQ ID No. 1.
30. An antibody according to claim 29, wherein the alanine scan is performed as described in example 16 herein or substantially as described in example 16 herein.
31. The antibody according to claims 29 to 30, wherein the alanine scan is performed by a procedure comprising the steps of:
i) expressing a mutant human 5T4 polypeptide and a wild type 5T4 polypeptide (amino acid residues 32-355 of SEQ ID NO: 1) individually in human embryonic kidney cells, such as HEK 293 cells, to provide a sample comprising 70-90.000 cells, such as 80.000 cells, for each mutant or wild type 5T4, in which mutant human 5T4 polypeptide all amino acid residues in the extracellular domain of human 5T4 (corresponding to amino acid residues 32-355 of SEQ ID NO: 1) are individually substituted with alanine, except cysteine and alanine,
ii) incubating the cells in each sample with 20 μ L of the antibody conjugated with Fluorescein Isothiocyanate (FITC) antibody (3 μ g/mL; in FACS buffer), then washing each sample twice in 150-,
iii) for each sample, determining the average amount of antibody bound per cell as the geometric mean of fluorescence intensity (gMFI) of the population of living single cells in the sample, and normalizing the data for each test antibody against the binding intensity of a non-cross-blocking 5T 4-specific control antibody using the following equation:
Figure FDA0002720035650000061
wherein "aa position" refers to a position mutated to alanine,
wherein a Z-score is calculated to express the loss or gain of antibody binding according to the following calculation:
Figure FDA0002720035650000062
where μ and σ are the mean and standard deviation, respectively, of the normalized gMFI calculated from all mutants,
wherein the gMFI of the control antibody is lower than the average gMFI for a particular 5T4 mutantControl antibody2.5 Xaverage gMFIControl antibodySD (from all mutants) of (c), data were excluded from the analysis; and optionally
Wherein data is excluded from the analysis if a residue binds with a Z-score just below-1.5 (e.g., -1.5 to-1.8, such as-1.5 to-1.7 or such as-1.5 to-1.6) and the residue is predicted to be buried and spatially separated from the majority of residues that are predicted to be surface exposed and for which loss of or reduced binding is determined.
32. The antibody according to claim 31, wherein the non-cross blocking 5T4 specific control antibody in step iv) is a bispecific antibody comprising
-an antigen binding region comprising the VH sequence shown as SEQ ID NO:83 and the VL sequence shown as SEQ ID NO:84 [ a1 ]; and
an antigen-binding region comprising the VH sequence shown as SEQ ID NO:97 and the VL sequence shown as SEQ ID NO:98 [ B12 ].
33. The antibody according to any one of claims 1 to 20, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues R73, Y92 and R94 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
34. The antibody according to any one of claims 1 to 20 and 33, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues S69, R73, Y92 and R94 is substituted with alanine, there is loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
35. The antibody according to any one of claims 1 to 20 and 33, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues R73, T74, Y92, R94 and N95 is substituted with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
36. The antibody according to any one of claims 1 to 20 and 33 to 35, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues L89, F111, L117, F138, L144, D148, N152 is substituted with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
37. The antibody according to any one of claims 1 to 20 and 33 to 36, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues R73, Y92, R94, F111, F138, L144 and D148 is substituted with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
38. The antibody according to any one of claims 1 to 20 and 33 to 36, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues S69, R73, Y92, R94, F111, F138 and D148 is substituted with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
39. The antibody according to any one of claims 1 to 20 and 33 to 36, wherein the antibody binds to 5T4 such that if any one or more of amino acid residues R73, T74, Y92, R94, N95 and F138 is substituted with alanine, there is a loss of binding or binding is reduced; the numbering of each amino acid residue refers to its position in SEQ ID NO 1.
40. The antibody according to any one of claims 33 to 39, wherein the effect of the alanine substitution is determined by an alanine scan of a polypeptide comprising amino acid residues 32-355 of SEQ ID NO 1.
41. The antibody according to any one of claims 33 to 39, wherein the effect of alanine substitution is determined by a procedure as described in example 16 herein or substantially as described in example 16 herein.
42. The antibody according to any one of claims 33 to 39, wherein loss of binding is defined as a Z-score of less than 1.5 for binding; the Z-score is optionally calculated as described in example 16 herein or substantially as described in example 16 herein.
43. The antibody according to claim 42, wherein the effect of the alanine substitution is determined by a procedure comprising the steps of:
i) expressing a mutant human 5T4 polypeptide and a wild-type 5T4 polypeptide individually in human embryonic kidney cells, such as HEK 293 cells, to provide a sample comprising 70-90.000 cells, such as 80.000 cells, for each mutant or wild-type 5T4, in which mutant human 5T4 polypeptide all amino acid residues in the human 5T4 extracellular domain (corresponding to amino acid residues 32-355 of SEQ ID NO: 1) are individually substituted with alanine, except cysteine and alanine,
ii) incubating the cells in each sample with 20 μ L of the antibody conjugated with Fluorescein Isothiocyanate (FITC) antibody (3 μ g/mL; in FACS buffer), then washing each sample twice in 150-,
iii) for each sample, determining the average amount of antibody bound per cell as the geometric mean of fluorescence intensity (gMFI) of the population of living single cells in the sample, and normalizing the data for each test antibody against the binding intensity of a non-cross-blocking 5T 4-specific control antibody using the following equation:
Figure FDA0002720035650000081
wherein "aa position" refers to a position mutated to alanine,
wherein a Z-score is calculated to express the loss or gain of antibody binding according to the following calculation:
Figure FDA0002720035650000082
where μ and σ are the mean and standard deviation, respectively, of the normalized gMFI calculated from all mutants,
wherein the gMFI of the control antibody is lower than the average gMFI for a particular 5T4 mutantControl antibody2.5 Xaverage gMFIControl antibodySD (from all mutants) of (c), data were excluded from the analysis; and optionally
Wherein data is excluded from the analysis if a residue binds with a Z-score just below-1.5 (e.g., -1.5 to-1.8, such as-1.5 to-1.7 or such as-1.5 to-1.6) and the residue is predicted to be buried and spatially separated from the majority of residues that are predicted to be surface exposed and for which loss of or reduced binding is determined.
44. The antibody according to claim 43, wherein the non-cross blocking 5T4 specific control antibody in step iii) is a bispecific antibody comprising
-an antigen binding region comprising the VH sequence shown as SEQ ID NO:83 and the VL sequence shown as SEQ ID NO:84 [ a1 ]; and
an antigen-binding region comprising the VH sequence shown as SEQ ID NO:97 and the VL sequence shown as SEQ ID NO:98 [ B12 ].
45. The antibody according to any one of the preceding claims, wherein the antibody has reduced internalization capability as shown by reduced cytotoxicity when conjugated to a cytotoxic moiety as compared to an antibody [ H8] comprising a heavy chain Variable (VH) region comprising the sequence shown as SEQ ID NO:87 and a light chain Variable (VL) region comprising the sequence shown as SEQ ID NO: 88.
46. The antibody according to claim 45, wherein cytotoxicity is determined using a procedure as described in example 7, e.g. in a procedure comprising the steps of:
i) Providing a monovalent antibody comprising a first Fab arm of an antibody as defined in any of the preceding claims, and a second Fab arm capable of binding to the HIV viral protein gp120(HIV-1gp120) and conjugated to Duostatin-3,
ii) contacting breast cancer cells MDA-MB-468(ATCC clone HTB-132) or HCC1954(ATCC clone CRL-1338) with said monovalent antibody at 37 ℃ for 5 days; and is
iii) determining the viability of said cells.
47. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8 [059],
b) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [076] of SEQ ID NOS 13, 14 and 15,
c) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 20, 21 and 22 [085],
d) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [106] of SEQ ID NOs: 27, 28 and 29,
e) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [127] of SEQ ID NOs: 34, 35 and 36,
f) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [207] of SEQ ID NOs: 41, 42 and 43,
g) A heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [226] of SEQ ID NOs: 48, 49 and 50, and
h) a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3 sequences collectively comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to g).
48. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8 [059],
b) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences [207] of SEQ ID NOs: 41, 42 and 43,
c) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 48, 49 and 50 [226 ]; and
d) a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3 sequences collectively comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to c).
49. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 6, 7 and 8 [059 ].
50. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of: heavy chain Variable (VH) region [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43.
51. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of: heavy chain Variable (VH) region [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS 48, 49 and 50.
52. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 6, 7 and 8, respectively, and a light chain Variable (VL) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 10, AAS and SEQ ID NO:11, respectively [059],
b) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 13, 14 and 15, respectively, and a light chain Variable (VL) region [076] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 17, DAS and SEQ ID NO:18, respectively,
c) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS 20, 21 and 22, respectively, and a light chain Variable (VL) region [085] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 24, DAS and SEQ ID NO 25, respectively,
d) A heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 27, 28 and 29, respectively, and a light chain Variable (VL) region [106] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 31, DVS and SEQ ID NOs 32, respectively,
e) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOS: 34, 35 and 36, respectively, and a light chain Variable (VL) region [127] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:38, DAS and SEQ ID NO:39, respectively,
f) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 41, 42 and 43, respectively, and a light chain Variable (VL) region [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 45, DAS and SEQ ID NO. 46, respectively,
g) a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 48, 49 and 50, respectively, and a light chain Variable (VL) region [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO. 52, DAS and SEQ ID NO. 53, respectively; and
h) a heavy chain Variable (VH) region and a light chain Variable (VL) region, each region comprising a CDR1, CDR2 and CDR3 sequence, said CDR1, CDR2 and CDR3 sequences collectively comprising at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or at most 10 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to g).
53. The antibody according to any one of the preceding claims, wherein the six Complementarity Determining Regions (CDRs) capable of binding the antigen binding region of 5T4 together comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or at most 10 amino acid substitutions when compared to;
iv) the CDR sequences of SEQ ID NO 6, 7, 8, 10, AAS and SEQ ID NO 11 [059],
v) CDR sequences of SEQ ID NO 41, 42, 43, 45, DAS and SEQ ID NO 46 [207 ]; or
vi) CDR sequences of SEQ ID NO 48, 49, 50, 52, DAS and SEQ ID NO 53 [226 ].
54. The antibody according to any one of claims 22 to 29, wherein 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 of said amino acid substitutions are conservative amino acid substitutions.
55. The antibody according to any one of the preceding claims, which antibody comprises one or two heavy chain variable regions, wherein complementarity determining region 3(CDR3) comprises six consecutive amino acid residues of the sequence shown in SEQ ID NO:102(YYGMDV) [059, 207, 226 ].
56. An antibody according to claim 55, wherein said six consecutive amino acid residues are the C-most amino acid residue within CDR 3.
57. The antibody of any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises one or two heavy chain Variable (VH) region comprising the CDR1 sequence of SEQ ID NO:41(GGSFSGYY), SEQ ID NO:103 (IDHSX) 1ST) and the CDR2 sequence of SEQ ID NO 104 (AX)2WFGELX3X4YYYGMDV) and the light chain variable region comprises the CDR3 sequence of SEQ ID NO 105 (QSVSSX)5) The CDR1 sequence, the CDR2 sequence DAS and the CDR3 sequence of SEQ ID NO:46(QQRSNWPLT) of (I), wherein X1Is G or E, X2Is A or G, X3Is W or Y, X4Is D or H, and X5Is Y or F [207, 226]。
58. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8, respectively, and a light chain Variable (VL) region [059] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 10, AAS and SEQ ID NOs 11, respectively.
59. An antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 41, 42 and 43 respectively and a light chain Variable (VL) region [207] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:45, DAS and SEQ ID NO:46 respectively.
60. An antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 48, 49 and 50 respectively and a light chain Variable (VL) region [226] comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NO 52, DAS and SEQ ID NO 53 respectively.
61. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:5 or a sequence [059] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:5,
b) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 12 [076],
c) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 19 or a sequence [085] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 19,
d) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 26 or a sequence [106] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 26,
e) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:33 or a sequence [127] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:33,
f) A heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:40 or a sequence [207] having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 40; and
g) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 [226 ].
62. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO. 5 or a sequence [059] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 5.
63. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:40 or a sequence [207] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 40.
64. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 47 or a sequence [226] having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 47.
65. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO. 5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 5 and a light chain Variable (VL) region [059] comprising the sequence of SEQ ID NO. 9 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 9,
b) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO. 12 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 12 and a light chain Variable (VL) region [076] comprising the sequence of SEQ ID NO. 16 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO. 16,
c) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 19 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 19 and a light chain Variable (VL) region [085] comprising the sequence of SEQ ID NO 23 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 23,
d) A heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 26 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 26 and a light chain Variable (VL) region [106] comprising the sequence of SEQ ID NO 30 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 30,
e) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 33 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 33 and a light chain Variable (VL) region [127] comprising the sequence of SEQ ID NO 37 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 37,
f) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 40 and a light chain Variable (VL) region [207] comprising the sequence of SEQ ID NO 44 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO 44,
g) A heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 and a light chain Variable (VL) region [226] comprising the sequence of SEQ ID NO:51 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 51.
66. The antibody according to any one of the preceding claims, wherein the antigen binding region capable of binding 5T4 comprises a heavy chain Variable (VH) region and a light chain Variable (VL) region selected from the group consisting of:
a) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO. 5 and a light chain Variable (VL) region comprising the sequence of SEQ ID NO. 9 [059],
b) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO. 12 and a light chain Variable (VL) region [076] comprising the sequence of SEQ ID NO. 16,
c) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 19 and a light chain Variable (VL) region [085] comprising the sequence of SEQ ID NO 23,
d) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 26 and a light chain Variable (VL) region [106] comprising the sequence of SEQ ID NO 30,
e) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO 33 and a light chain Variable (VL) region [127] comprising the sequence of SEQ ID NO 37,
f) A heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:40 and a light chain Variable (VL) region [207] comprising the sequence of SEQ ID NO: 44; and
g) a heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:47 and a light chain Variable (VL) region comprising the sequence of SEQ ID NO:51 [226 ].
67. The antibody according to any one of the preceding claims, wherein the antibody is a full length antibody, such as a full length IgG1 antibody.
68. The antibody according to any one of the preceding claims, which is a monovalent antibody.
69. The antibody according to any one of the preceding claims, which is a bivalent antibody.
70. The antibody according to any one of the preceding claims, which is a monospecific antibody.
71. The antibody according to any one of the preceding claims, which is a bispecific antibody.
72. The antibody according to any one of the preceding claims, comprising an antigen binding region of an antibody that binds CD3, such as human CD3(epsilon), such as human CD3(epsilon) as specified in SEQ ID NO: 4.
73. The antibody of claim 72, wherein the antigen binding region that binds CD3 comprises
A heavy chain Variable (VH) region comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 54, 55 and 56, respectively; [ wild-type anti-CD 3(SP 34/humanized SP34, WO2015001085(Genmab)) -VH CDR sequences ];
And optionally
A light chain Variable (VL) region comprising the CDR1, CDR2 and CDR3 sequences [ wild type anti-CD 3, VL CDR sequences ] of SEQ ID NOs: 58, GTN and 59, respectively.
74. An antibody according to claim 72 or 73, wherein said antigen binding region that binds CD3 comprises
A heavy chain Variable (VH) region comprising the sequence of SEQ ID NO:57 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:57 [ wild-type anti-CD 3-VH full-length sequence ];
and optionally
A light chain Variable (VL) region comprising the sequence of SEQ ID NO:60 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:60 [ wild-type anti-CD 3-VL full length sequence ].
75. The antibody according to any one of claims 72 to 74, wherein the antibody has a lower binding affinity for human CD3 than an antibody having an antigen-binding region comprising the VH sequence shown in SEQ ID NO:57 and the VL sequence shown in SEQ ID NO:60 [ wild-type anti-CD 3 (humanized SP34, WO2015001085(Genmab)) VH and VL sequences ], preferably wherein the affinity is at least 5-fold lower, such as at least 10-fold lower, for example at least 20-fold lower, at least 30-fold lower, at least 40-fold lower, at least 45-fold lower or such as at least 50-fold lower.
76. An antibody according to any one of claims 72 to 74Wherein the antigen-binding region has an equilibrium dissociation constant K of 200-1000 nM, such as 300-1000 nM, 400-1000 nM, 500-1000 nM, 300-900 nM, 400-700 nM, 500-900 nM, 500-800 nM, 500-700 nM, 600-1000 nM, 600-900 nM, 600-800 nM, or 600-700 nMDBinds to CD 3.
77. An antibody according to claim 72 or 75, wherein the antigen-binding region has an equilibrium constant K in the range of 1-100 nM, such as in the range of 5-100 nM, in the range of 10-100 nM, in the range of 1-80 nM, in the range of 1-40 nM in the range of 1-60 nM, in the range of 1-20 nM, in the range of 5-80 nM, in the range of 5-60 nM, in the range of 5-40 nM, in the range of 5-20 nM, in the range of 10-80 nM, in the range of 10-60 nM, in the range of 10-40 nM, or as in the range of 10-20 nM dissociationDBinds to CD 3.
78. The antibody according to any one of claims 72 to 77, wherein
The antigen binding region that binds CD3 comprises a heavy chain Variable (VH) region comprising a CDR1 sequence, a CDR2 sequence and a CDR3 sequence,
When compared to a heavy chain Variable (VH) region comprising the sequence set forth in SEQ ID NO:57, the heavy chain Variable (VH) region has an amino acid substitution in one of the CDR sequences, the substitution being at a position selected from the group consisting of: t31, N57, H101, G105, S110 and Y114, the positions being numbered according to the sequence of SEQ ID NO:57[ VH _ huCD3-H1L1 ]; and
the wild-type light chain Variable (VL) region comprises the CDR1, CDR2 and CDR3 sequences shown as SEQ ID NOS 58, GTN and SEQ ID NO 59, respectively.
79. The antibody according to claim 72, wherein the CDR1, CDR2 and CDR3 of the heavy chain Variable (VH) region that binds the antigen binding region of CD3 collectively comprise up to 1, 2, 3, 4 or 5 amino acid substitutions when compared to the CDR1, CDR2 and CDR3 of the sequence set forth in SEQ ID NO. 57.
80. The antibody according to claim 72 or 73, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain Variable (VH) region that binds to the antigen binding region of CD3 have at least 95% sequence identity, such as at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity or at least 99% sequence identity, with the amino acid sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain Variable (VH) region, the sequence identity being calculated based on aligning the amino acid sequence consisting of the sequences of CDR1, CDR2 and CDR3 of the heavy chain Variable (VH) region of the antigen binding region that binds CD with the amino acid sequences comprising the sequences of CDR1, CDR2 and CDR3 of the wild-type heavy chain Variable (VH) region.
81. The antibody according to claims 72 to 74, wherein the antigen binding region that binds CD3 comprises a mutation selected from the group consisting of: T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R, Y114V.
82. The antibody according to any one of the preceding claims, wherein when the antibody is a bispecific antibody that lacks Fc-mediated effector function or has reduced Fc-mediated effector function ("inert" antibody), and comprises the antigen binding region of an antibody that binds CD3, then the antibody:
a) when purified PBMC or T cells are used as effector cells, it is possible to mediate concentration-dependent cytotoxicity of SK-OV-3 cells, e.g., when assayed as described in example 14 herein,
b) when purified T cells are used as effector cells, it is possible to mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, e.g., when assayed as described in example 13 herein,
c) capable of activating T cells in vitro in the presence of MDA-MB-231 tumor cells; for example when measured as described in example 13 herein,
d) capable of activating T cells in vitro in the presence of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells; for example when measured as described in example 17 herein,
e) When purified T cells are used as effector cells, capable of inducing cytotoxicity of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells, e.g., when assayed as described in example 17 herein; and/or
f) Reconstitution of mouse xenograft models in humanized immune hematopoietic stem cells, such as NOD. Cg-Prkdc inoculated with human MDA-MB-231 tumor cellsscidIl2rgtm1WjlAnti-tumor activity, such as delayed tumor outgrowth, is shown in/SzJ; for example when measured as described in example 15.
83. An antibody according to claim 82, wherein the ability of the antibody to mediate concentration-dependent cellular cytotoxicity of SK-OV-3 cells is determined in an in vitro cytotoxicity assay comprising the steps of:
i) isolating Peripheral Blood Mononuclear Cells (PBMC) or T cells from a buffy coat of a healthy human donor,
ii) providing
A first set of samples, wherein each sample comprises PBMCs and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of PBMCs to SK-OV-3 cells in the sample is 1:2,1:1,2:1,4:1,8:1, and 12: 1; and
a second set of samples, wherein each sample comprises T cells and human ovarian adenocarcinoma SK-OV-3 cells, and wherein the ratio of T cells to SK-OV-3 cells in the sample is 1:2,1:1,2:1,4:1, and 8:1
iii) adding the antibody to each set of samples at a concentration ranging from 0.0128ng/mL to 1000ng/mL and incubating the samples at 37 ℃ for 72 hours; then the
iv) viability of the SK-OV-3 cells was assessed using Resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide).
84. The antibody of claim 82, wherein the ability to activate T cells in vitro in the presence of MDA-MB-231 tumor cells is determined in an assay comprising the steps of:
i) isolating T cells from a healthy human donor buffy coat,
ii) providing a set of samples, wherein each sample comprises T cells and human breast cancer MDA-MB-231 cells, and wherein the ratio of T cells to MDA-MB-231 cells in the sample is 8:1,
iii) adding said antibody to the set of samples at a concentration ranging from 0.0128ng/mL to 1000ng/mL and incubating said samples at 37 ℃ for 72 hours,
iv) staining the T cells with fluorescently labeled antibodies against T cell activation markers, such as CD69-APC, CD25-PE-Cy7 and CD279/PD1-BV604 antibodies, by incubation with the antibodies at 4 ℃ for 30 minutes; and is
v) analyzing the sample by flow cytometry.
85. The antibody of claim 82, wherein T cells are activated in vitro in the presence of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells as determined by a procedure comprising:
i) Providing T cells isolated from a healthy human donor buffy coat,
ii) providing a set of samples, wherein each sample comprises said T cells and BxPC-3, PANC-1, Ca Ski or SiHa tumor cells, and wherein the ratio of T cells to tumor cells in said sample is 4:1,
iii) adding the antibody to the set of samples at a concentration ranging from 0.0128ng/mL to 5000ng/mL (e.g., a 5-fold dilution), and incubating the samples at 37 ℃ for 72 hours,
iv) collecting 110 μ L of T cell containing supernatant from each sample and staining the T cells with fluorescently labeled antibodies to T cell markers, such as CD3-eFluor450, CD4-APC-eFluor780, DC8-AF700, and with antibodies to T cell markers, such as 69-APC, CD25-PE-Cy7 and CD279/PD1-BV604 antibodies, by incubation with the antibodies at 4 ℃ for 30 minutes; and is
v) analyzing the sample by flow cytometry.
86. The antibody of claim 82, wherein the ability to induce cytotoxicity of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells is determined by a procedure comprising the steps of:
i) providing T cells isolated from a healthy human donor buffy coat,
ii) providing a set of test and control samples, wherein each sample comprises BxPC-3, PANC-1, Ca Ski or SiHa tumor cells and the T cells that have been allowed to adhere to the bottom of a 96-well tissue culture plate, and wherein the ratio of T cells to tumor cells in the sample is 4:1,
iii) adding the antibody to the test sample set at a concentration ranging from 0.0128ng/mL to 5000ng/mL (e.g., a 5-fold dilution) while the control sample remains untreated or is incubated with 5. mu.M staurosporine, then incubating all samples at 37 ℃ for 72 hours,
iv) incubation of adherent cells in 10% (w/w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide (Resazurin) in RPMI-1640 medium supplemented with 10% (w/w) donor bovine serum containing iron and penicillin/streptomycin at 37 ℃ for 4 hours,
v) measuring the absorbance of the cells; the absorbance of cells incubated with staurosporine was set to 0% viability and the absorbance of untreated cells was set to 100% viability and the percentage of viable cells was calculated as follows:
Figure FDA0002720035650000201
87. the antibody according to any one of claims 72 and 78 to 84, wherein the antigen binding region capable of binding CD3 comprises:
a) heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR1-T31P + wild type VH CDR2, 3] having the sequences shown in SEQ ID NO:61, 55 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
b) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR1-T31M + wild type VH CDR2, 3] having the sequences shown in SEQ ID NO:63, 55 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
c) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ VH CDR-N57E + wild type VH CDR1, 3] having the sequences shown in SEQ ID NO:54, 65 and 56, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
d) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown in SEQ ID NO:54, 55 and 67, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
e) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101N ] having the sequences shown in SEQ ID NO:54, 55 and 69, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
f) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-G105P ] having the sequences shown in SEQ ID NO:54, 55 and 71, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
g) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-S110A ] having the sequences shown in SEQ ID NO:54, 55 and 73, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
h) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-S110G ] having the sequences shown in SEQ ID NO:54, 55 and 75, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
i) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114V ] having the sequences shown in SEQ ID NO:54, 55 and 77, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
j) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114M ] having the sequences shown in SEQ ID NO:54, 55 and 79, respectively, and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown in SEQ ID NO:58, GTN and SEQ ID NO:59, respectively, or
k) Heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-Y114R ] having the sequences shown in SEQ ID NO:54, 55 and 81 respectively and light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequence shown in SEQ ID NO:58, sequence GTN and sequence shown in SEQ ID NO:59 respectively.
88. The antibody according to any one of claims 72 and 78 to 85, wherein the antigen binding region capable of binding CD3 comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID NOs 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, GTN and SEQ ID No. 59, respectively.
89. The antibody according to any one of claims 72 and 78 to 85, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 6, 7 and 8 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 10, AAS and SEQ ID NOs 11 respectively [059],
and
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
90. The antibody according to any one of claims 72 and 78 to 85, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 41, 42 and 43 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences [207] of SEQ ID NOs 45, DAS and SEQ ID NOs 46 respectively,
And
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
91. The antibody according to any one of claims 47 and 51 to 59, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 48, 49 and 50 respectively, and a light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences of SEQ ID NOs 52, DAS and SEQ ID NOs 53 respectively [207],
and
the antigen binding region capable of binding CD3 comprises a heavy chain Variable (VH) region comprising CDR1, CDR2 and CDR3[ wild type VH CDR1, 2+ VH CDR3-H101G ] having the sequences shown as SEQ ID nos. 54, 55 and 67, respectively, and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3[ wild type VL CDR1, 2, 3] having the sequences shown as SEQ ID No. 58, sequence GTN and SEQ ID No. 59, respectively.
92. The antibody of any one of claims 72 and 78 to 91, wherein the antigen-binding region capable of binding to human CD3 comprises a VH sequence and a VL sequence selected from the group consisting of SEQ ID NO:
a) a VH sequence [ VH T31P full-length sequence ] shown by SEQ ID NO:62 and a VL sequence [ wild type full-length sequence ] shown by SEQ ID NO:60,
b) a VH sequence [ VH T31M full-length sequence ] shown by SEQ ID NO:64 and a VL sequence shown by SEQ ID NO:60,
c) a VH sequence [ VH N57E full-length sequence ] shown by SEQ ID NO:66 and a VL sequence shown by SEQ ID NO:60,
d) a VH sequence [ VH H101G full-length sequence ] shown by SEQ ID NO:68 and a VL sequence shown by SEQ ID NO:60,
e) a VH sequence [ VH H101N full-length sequence ] shown by SEQ ID NO:70 and a VL sequence shown by SEQ ID NO:60,
f) a VH sequence [ VH G105P full-length sequence ] shown by SEQ ID NO:72 and a VL sequence shown by SEQ ID NO:60,
g) a VH sequence [ VH S110A full-length sequence ] shown by SEQ ID NO:74 and a VL sequence shown by SEQ ID NO:60,
h) a VH sequence [ VH S110G full-length sequence ] shown by SEQ ID NO:76 and a VL sequence shown by SEQ ID NO:60,
i) VH sequence [ VH Y114V full length sequence ] shown by SEQ ID NO:78 and VL sequence shown by SEQ ID NO:60,
j) a VH sequence shown as SEQ ID NO:80 [ VH Y114M full-length sequence ] and a VL sequence shown as SEQ ID NO: 60; and
k) VH sequence [ VH Y114R full length sequence ] shown as SEQ ID NO:82 and VL sequence shown as SEQ ID NO: 60.
93. The antibody according to any one of claims 72 and 78 to 92, wherein the antigen-binding region capable of binding to human CD3 comprises the VH sequence shown as SEQ ID NO:68 [ VH H101G full length sequence ] and the VL sequence shown as SEQ ID NO: 60.
94. The antibody according to any one of claims 78 to 93, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:5 [059-VH full length sequence ];
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
95. The antibody of any one of claims 72 and 78 to 93, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:40 [207-VH full length sequence ]; and
The antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
96. The antibody according to any one of claims 47 and 51 to 64, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47 [226-VH full length sequence ]; and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
97. The antibody of any one of claims 72 and 78 to 94, wherein
The antigen binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:5 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:5 [059 ];
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
98. The antibody of any one of claims 72, 78 to 93 and 95, wherein
The antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:40 and a heavy chain light region (VL) [ 207-VH + VL full length sequence ] comprising the sequence of SEQ ID NO:44 or a sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 44;
and
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
99. The antibody of any one of claims 72, 78 to 93 and 96, wherein
The antigen-binding region capable of binding 5T4 comprises a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:47 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO:47, and a heavy chain light region (VL) [ 226-VH + VL full-length sequence ], comprising the sequence of SEQ ID NO:51 or a sequence having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the sequence of SEQ ID NO: 51;
And
the antigen binding region capable of binding to human CD3 comprises a VH sequence shown as SEQ ID NO:68 [ VH H101G full-length sequence ] and a VL sequence shown as SEQ ID NO: 60.
100. An antibody according to any one of the preceding claims, wherein each antigen binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein the variable regions each comprise three CDR sequences, CDR1, CDR2 and CDR3 respectively, and four framework sequences, FR1, FR2, FR3 and FR4 respectively.
101. The antibody according to claim 100, wherein said antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).
102. The antibody according to claim 100 or 101, wherein said antibody comprises a first and a second heavy chain each comprising at least a hinge region, a CH2 and a CH3 region, wherein in said first heavy chain at least one amino acid in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted and in said second heavy chain at least one amino acid in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407 and K409 in the human IgG1 heavy chain has been substituted, wherein said substitutions of said first and said second heavy chains are not in the same position, and wherein said amino acid positions are numbered according to EU numbering.
103. The antibody according to any one of claims 100 to 102, wherein the amino acid in the position corresponding to K409 in the heavy chain of human IgG1 is R in the first heavy chain and the amino acid in the position corresponding to F405 in the heavy chain of human IgG1 is L in the second heavy chain, or vice versa.
104. The antibody according to any one of the preceding claims, wherein the antibody comprises first and second heavy chains, and wherein in both the first and second heavy chains the amino acid residues at positions corresponding to positions L234 and L235 in the heavy chain of human IgG1 according to EU numbering are F and E, respectively.
105. The antibody according to any one of the preceding claims, wherein the antibody comprises first and second heavy chains, and wherein in both the first and second heavy chains the amino acid residue at a position corresponding to position D265 in the heavy chain of human IgG1 according to EU numbering is a.
106. The antibody according to any one of the preceding claims, wherein
a) The antigen binding region capable of binding 5T4 is humanized, and/or
b) If present, the antigen binding region capable of binding to CD3 is humanized.
107. The antibody according to any one of the preceding claims, wherein
a) The antigen binding region capable of binding 5T4 is human, and/or
b) If present, the antigen binding region capable of binding CD3 is human.
108. The antibody according to any one of the preceding claims, wherein
a) The antigen binding region capable of binding 5T4 is chimeric, and/or
b) If present, the antigen binding region capable of binding CD3 is chimeric.
109. The antibody according to any one of the preceding claims, wherein the antibody comprises a first heavy chain and optionally a second heavy chain, and wherein the first heavy chain and, if present, the second heavy chain are modified such that the antibody induces Fc-mediated effector function to a lesser extent relative to the same unmodified antibody.
110. The antibody according to any one of the preceding claims, wherein the antibody comprises a kappa (kappa) light chain.
111. The antibody according to any one of the preceding claims, wherein the antibody comprises a lambda (λ) light chain.
112. The antibody according to any one of the preceding claims, wherein the antibody comprises a lambda (λ) light chain and a kappa (κ) light chain; for example, an antibody having a heavy chain comprising a binding region capable of binding CD3 and a lambda light chain; and a heavy chain and a kappa light chain comprising a binding region capable of binding 5T 4.
113. An immunoconjugate or antibody-drug conjugate (ADC) comprising an antibody according to any one of the preceding claims, and a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic drug, a cytokine, an immunosuppressive agent, an antibiotic, or a radioisotope.
114. A nucleic acid construct comprising
a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined in any one of claims 1 to 113, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined in any one of claims 72 to 113.
115. The nucleic acid construct according to claim 114, further comprising
a) Nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 113, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 113.
116. An expression vector comprising
a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined in any one of claims 1 to 112, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding 5T4 as defined in any one of claims 1 to 112.
117. The expression vector according to claim 116, further comprising
a) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 112, and/or
b) A nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 112.
118. A cell comprising a nucleic acid construct as defined in any of claims 114 to 115 or an expression vector as defined in claim 116 or 117, such as a cell, such as a recombinant host cell, which has been obtained by transfecting a host cell with said nucleic acid construct or expression vector.
119. The cell according to claim 118, wherein the host cell is of human origin, such as a Human Embryonic Kidney (HEK) cell, such as a HEK/Expi cell, or of rodent origin, such as a chinese hamster ovary cell, such as a CHO/N50 cell.
120. A composition comprising an antibody as defined in any one of claims 1 to 112.
121. A pharmaceutical composition comprising an antibody as defined in any one of claims 1 to 112 and a pharmaceutically acceptable carrier.
122. An antibody as defined in any one of claims 1 to 112 for use as a medicament.
123. The antibody of any one of claims 1 to 112 for use in the treatment of a disease.
124. The antibody for use according to claim 122 or 123, wherein the disease is cancer.
125. The antibody for use according to claim 124, wherein the cancer is characterized by expression of 5T4 in at least some tumor cells.
126. The antibody for use according to any one of claims 122 to 125, wherein the cancer is selected from the group consisting of: kidney/kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterus/endometrium/cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia.
127. A method of treating a disease, the method comprising administering to a subject in need thereof an antibody as defined in any one of claims 1 to 112, a composition as defined in claim 120 or a pharmaceutical composition as defined in claim 121.
128. A method according to claim 127, for treating cancer.
129. The method of claim 128, wherein the cancer is selected from the group consisting of: kidney/kidney cancer, breast cancer, colorectal cancer, prostate cancer, ovarian cancer, bladder cancer, uterus/endometrium/cervical cancer, lung cancer, gastrointestinal cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and neck cancer, lymphoma, acute myeloid leukemia.
130. A method for producing an antibody as defined in any one of claims 1 to 112, comprising the steps of:
a) culturing a host cell comprising an expression vector as defined in claim 114 or 117; and is
b) Purifying the antibody from the culture medium.
131. A method for producing an antibody as defined in any one of claims 1 to 112, comprising the steps of:
a) providing an antibody capable of binding 5T4 by culturing a host cell comprising an expression vector as defined in claim 116 or 117 under conditions allowing expression of the antibody capable of binding 5T4 and purifying the antibody capable of binding 5T4 from the culture medium;
b) providing an antibody capable of binding to CD3 by culturing a host cell comprising an expression vector comprising an antibody capable of binding to CD3 under conditions that allow expression of the antibody capable of binding to CD3, and purifying the antibody capable of binding to CD3 from the culture medium
I) A nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 112, and
II) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen binding region capable of binding CD3 as defined in any one of claims 72 to 112;
c) incubating the antibody capable of binding 5T4 with the antibody capable of binding CD3 under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and
d) obtaining the antibody.
132. A kit, such as for use as a companion diagnosis/for identifying within a patient population those patients who have a predisposition to respond to treatment with an antibody as defined in any one of claims 1 to 112 or an immunoconjugate or antibody-drug conjugate (ADC) as defined in claim 113, or for predicting the efficacy of said antibody or immunoconjugate or ADC when used to treat patients, comprising an antibody as defined in any one of claims 1 to 112; and instructions for use of the kit.
133. An anti-idiotypic antibody which binds to an antigen binding region capable of binding to 5T4 as defined in any one of claims 1 to 112.
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